neutravidin  (Thermo Fisher)


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    Structured Review

    Thermo Fisher neutravidin
    Course of the in vitro selection. As a measure for the enrichment of biotinylated d -glucagon-binding DNA sequences, the ratio of the fraction of library bound to the target immobilized on streptavidin or <t>neutravidin</t> beads versus the applied biotinylated peptide ( d -glucagon) concentration is plotted against the selection round numbers. A steep increase in binding is visible in rounds 6 to 8 followed by stagnation in rounds 9 to 12. In the following rounds the regular alternation between mutagenic and standard PCR results in alternating ratios.
    Neutravidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "A Mixed Mirror-image DNA/RNA Aptamer Inhibits Glucagon and Acutely Improves Glucose Tolerance in Models of Type 1 and Type 2 Diabetes *"

    Article Title: A Mixed Mirror-image DNA/RNA Aptamer Inhibits Glucagon and Acutely Improves Glucose Tolerance in Models of Type 1 and Type 2 Diabetes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.444414

    Course of the in vitro selection. As a measure for the enrichment of biotinylated d -glucagon-binding DNA sequences, the ratio of the fraction of library bound to the target immobilized on streptavidin or neutravidin beads versus the applied biotinylated peptide ( d -glucagon) concentration is plotted against the selection round numbers. A steep increase in binding is visible in rounds 6 to 8 followed by stagnation in rounds 9 to 12. In the following rounds the regular alternation between mutagenic and standard PCR results in alternating ratios.
    Figure Legend Snippet: Course of the in vitro selection. As a measure for the enrichment of biotinylated d -glucagon-binding DNA sequences, the ratio of the fraction of library bound to the target immobilized on streptavidin or neutravidin beads versus the applied biotinylated peptide ( d -glucagon) concentration is plotted against the selection round numbers. A steep increase in binding is visible in rounds 6 to 8 followed by stagnation in rounds 9 to 12. In the following rounds the regular alternation between mutagenic and standard PCR results in alternating ratios.

    Techniques Used: In Vitro, Selection, Binding Assay, Concentration Assay, Polymerase Chain Reaction

    2) Product Images from "Chemoenzymatic Site-Specific Labeling of Influenza Glycoproteins as a Tool to Observe Virus Budding in Real Time"

    Article Title: Chemoenzymatic Site-Specific Labeling of Influenza Glycoproteins as a Tool to Observe Virus Budding in Real Time

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002604

    Site-specifically labeled HA-Srt protein is incorporated into virions. ( A ) Experimental setup. Confluent monolayers of MDCK cells were infected with an MOI = 0.5 during 4.5 hours after which cells were starved and pulse-labeled with [ S 35]Cysteine/Methionine for 20 minutes. After a 2 hour chase, a second pulse-labeling was performed using 100 µM sortase and 250 µM biotin probe to label surface accessible HA-Srt. At indicated timepoints, both cell supernatant as well as cell lysate was analyzed for presence of viral proteins. ( B ) Surface behavior HA on infected MDCK cells analyzed via affinity adsorption to neutravidin-agarose. At indicated timepoint, cells were lysed in 0.5% NP40 buffer and biotin labeled HA-Srt remaining at the cell surface recovered via affinity adsorption on neutravidin-agarose. Proteins were eluted with 2× SDS sample buffer, resolved by 12.5% SDS-PAGE and visualized via autoradiography. ( C ) Accumulation of HA-biotin in supernatant analyzed by affinity adsorption to neutravidin-agarose. Accumulation of biotin-HA-Srt in the supernatant of the cells analyzed in 4B was measured via immunoprecipitation on neutravidin-agarose. Supernatant was lysed via addition of NP40 buffer prior to biding to beads. Proteins were eluted with 2× SDS sample buffer, resolved by 12.5% SDS-PAGE and visualized via autoradiography. ( D ) Quantification of HA loss from the cell surface. Densitometric quantification of radioactivity was performed on autoradiographs from figure 4B and 4C . Total levels of HA-Srt were quantified relative to the levels at the cell surface at t = 0 hrs (top graph). To quantify the kinetics of budding, loss of HA-Srt from the cell surface was quantified as percent reduction relative to the t = 0 timepoint at the cell surface. The rate of accumulation in the cell supernatant was quantified relative to the maximal amount recovered at the t = 10 hrs timepoint. ( E ) Accumulation of whole virus particles analyzed by affinity adsorption to chicken erythrocytes. Accumulation of complete virus particles in the cell supernatant was measured via affinity adsorption on chicken erythrocytes. Supernatant from cells analyzed in 4B was removed at indicated timepoints and mixed with chicken erythrocytes for 30 minutes at 4°C. Cells and bound viral particles were lysed in 2× SDS sample buffer, proteins resolved on 12.5% SDS-PAGE and visualized via autoradiography. ( F ) Kinetcs of virus accumulation as analyzed by adsorption to neutravidin-agarose versus erythrocytes. Densitometric quantification of radioactivity was performed to compare the rate of HA-Srt accumulation in the supernatant compared to whole viral particles (4C versus 4E). Numbers were normalized at t = 1 hrs at which both methods were used.
    Figure Legend Snippet: Site-specifically labeled HA-Srt protein is incorporated into virions. ( A ) Experimental setup. Confluent monolayers of MDCK cells were infected with an MOI = 0.5 during 4.5 hours after which cells were starved and pulse-labeled with [ S 35]Cysteine/Methionine for 20 minutes. After a 2 hour chase, a second pulse-labeling was performed using 100 µM sortase and 250 µM biotin probe to label surface accessible HA-Srt. At indicated timepoints, both cell supernatant as well as cell lysate was analyzed for presence of viral proteins. ( B ) Surface behavior HA on infected MDCK cells analyzed via affinity adsorption to neutravidin-agarose. At indicated timepoint, cells were lysed in 0.5% NP40 buffer and biotin labeled HA-Srt remaining at the cell surface recovered via affinity adsorption on neutravidin-agarose. Proteins were eluted with 2× SDS sample buffer, resolved by 12.5% SDS-PAGE and visualized via autoradiography. ( C ) Accumulation of HA-biotin in supernatant analyzed by affinity adsorption to neutravidin-agarose. Accumulation of biotin-HA-Srt in the supernatant of the cells analyzed in 4B was measured via immunoprecipitation on neutravidin-agarose. Supernatant was lysed via addition of NP40 buffer prior to biding to beads. Proteins were eluted with 2× SDS sample buffer, resolved by 12.5% SDS-PAGE and visualized via autoradiography. ( D ) Quantification of HA loss from the cell surface. Densitometric quantification of radioactivity was performed on autoradiographs from figure 4B and 4C . Total levels of HA-Srt were quantified relative to the levels at the cell surface at t = 0 hrs (top graph). To quantify the kinetics of budding, loss of HA-Srt from the cell surface was quantified as percent reduction relative to the t = 0 timepoint at the cell surface. The rate of accumulation in the cell supernatant was quantified relative to the maximal amount recovered at the t = 10 hrs timepoint. ( E ) Accumulation of whole virus particles analyzed by affinity adsorption to chicken erythrocytes. Accumulation of complete virus particles in the cell supernatant was measured via affinity adsorption on chicken erythrocytes. Supernatant from cells analyzed in 4B was removed at indicated timepoints and mixed with chicken erythrocytes for 30 minutes at 4°C. Cells and bound viral particles were lysed in 2× SDS sample buffer, proteins resolved on 12.5% SDS-PAGE and visualized via autoradiography. ( F ) Kinetcs of virus accumulation as analyzed by adsorption to neutravidin-agarose versus erythrocytes. Densitometric quantification of radioactivity was performed to compare the rate of HA-Srt accumulation in the supernatant compared to whole viral particles (4C versus 4E). Numbers were normalized at t = 1 hrs at which both methods were used.

    Techniques Used: Labeling, Infection, Adsorption, SDS Page, Autoradiography, Immunoprecipitation, Radioactivity

    3) Product Images from "Surface Functionalization Methods to Enhance Bioconjugation in Metal-Labeled Polystyrene Particles"

    Article Title: Surface Functionalization Methods to Enhance Bioconjugation in Metal-Labeled Polystyrene Particles

    Journal: Macromolecules

    doi: 10.1021/ma200582q

    The bioconjugation conditions used to attach NeutrAvidin to the particle surface
    Figure Legend Snippet: The bioconjugation conditions used to attach NeutrAvidin to the particle surface

    Techniques Used:

    4) Product Images from "Avidin-Based Targeting and Purification of a Protein IX-Modified, Metabolically Biotinylated Adenoviral Vector"

    Article Title: Avidin-Based Targeting and Purification of a Protein IX-Modified, Metabolically Biotinylated Adenoviral Vector

    Journal: Molecular therapy : the journal of the American Society of Gene Therapy

    doi: 10.1016/j.ymthe.2004.03.006

    BAP-modified vectors are metabolically biotinylated and display biotins on their surface. (A) Western blot of vectors. 5 × 10 8 viral particles were boiled in Laemmli loading buffer and loaded onto a 7.5% polyacrylamide gel. Separated proteins were blotted onto PVDF membranes and probed with rabbit anti-[Ad5] polyclonal antibody (loading control) or neutravidin – HRP. (B) Magnetic µparticle capture assay. Vectors were incubated with streptavidin-coated paramagnetic µparticles and run through a MACS magnetic column as described under Materials and Methods. Samples were assayed for transduction on HeLa cells by flow cytometry and percentage transduction (output/input) was calculated for each vector. Results are presented as means ± standard deviation of three experiments. (C) Surface biotinylation ELISA. Serial dilutions of BAP-modified or wild-type vectors were adsorbed onto wells of an ELISA plate. Wells were blocked, washed, and probed with neutravidin – HRP. Results are presented as means ± standard deviation of three experiments.
    Figure Legend Snippet: BAP-modified vectors are metabolically biotinylated and display biotins on their surface. (A) Western blot of vectors. 5 × 10 8 viral particles were boiled in Laemmli loading buffer and loaded onto a 7.5% polyacrylamide gel. Separated proteins were blotted onto PVDF membranes and probed with rabbit anti-[Ad5] polyclonal antibody (loading control) or neutravidin – HRP. (B) Magnetic µparticle capture assay. Vectors were incubated with streptavidin-coated paramagnetic µparticles and run through a MACS magnetic column as described under Materials and Methods. Samples were assayed for transduction on HeLa cells by flow cytometry and percentage transduction (output/input) was calculated for each vector. Results are presented as means ± standard deviation of three experiments. (C) Surface biotinylation ELISA. Serial dilutions of BAP-modified or wild-type vectors were adsorbed onto wells of an ELISA plate. Wells were blocked, washed, and probed with neutravidin – HRP. Results are presented as means ± standard deviation of three experiments.

    Techniques Used: Modification, Metabolic Labelling, Western Blot, Incubation, Magnetic Cell Separation, Transduction, Flow Cytometry, Cytometry, Plasmid Preparation, Standard Deviation, Enzyme-linked Immunosorbent Assay

    Redirection of Ad-IX-BAP tropism with biotinylated ligands. (A) Transduction of CAR-negative K562 cells with biotinylated antibodies. Cells were incubated with biotinylated anti-[CD59] antibody, washed, saturated with neutravidin, washed, and incubated with viral vectors. Controls were incubated with antibody or avidin alone, followed by vector. Transduction efficiency was measured by flow cytometry 48 h posttransduction. (B) Transduction of chemically biotinylated K562 cells. Cell surface proteins of K562 cells were chemically biotinylated with NHS-LC-biotin. Cells were then saturated with neutravidin, washed, and incubated with vector. Controls include treatment with NHS-biotin or neutravidin alone, followed by vector. Transduction efficiency was measured by flow cytometry 96 h posttransduction. (C) Transduction of murine C 2 C 12 myotubes with biotinylated transferrin. Cells were incubated with biotinylated transferrin, washed, bound with excess neutravidin, washed, and incubated with viral vectors. Controls were incubated with buffer or avidin alone, followed by vector. Cells were photographed under a fluorescence microscope 36 h posttransduction.
    Figure Legend Snippet: Redirection of Ad-IX-BAP tropism with biotinylated ligands. (A) Transduction of CAR-negative K562 cells with biotinylated antibodies. Cells were incubated with biotinylated anti-[CD59] antibody, washed, saturated with neutravidin, washed, and incubated with viral vectors. Controls were incubated with antibody or avidin alone, followed by vector. Transduction efficiency was measured by flow cytometry 48 h posttransduction. (B) Transduction of chemically biotinylated K562 cells. Cell surface proteins of K562 cells were chemically biotinylated with NHS-LC-biotin. Cells were then saturated with neutravidin, washed, and incubated with vector. Controls include treatment with NHS-biotin or neutravidin alone, followed by vector. Transduction efficiency was measured by flow cytometry 96 h posttransduction. (C) Transduction of murine C 2 C 12 myotubes with biotinylated transferrin. Cells were incubated with biotinylated transferrin, washed, bound with excess neutravidin, washed, and incubated with viral vectors. Controls were incubated with buffer or avidin alone, followed by vector. Cells were photographed under a fluorescence microscope 36 h posttransduction.

    Techniques Used: Transduction, Incubation, Avidin-Biotin Assay, Plasmid Preparation, Flow Cytometry, Cytometry, Fluorescence, Microscopy

    5) Product Images from "Single Vesicle Assaying of SNARE-Synaptotagmin-Driven Fusion Reveals Fast and Slow Modes of Both Docking and Fusion and Intrasample Heterogeneity"

    Article Title: Single Vesicle Assaying of SNARE-Synaptotagmin-Driven Fusion Reveals Fast and Slow Modes of Both Docking and Fusion and Intrasample Heterogeneity

    Journal: Biophysical Journal

    doi: 10.1016/j.bpj.2010.12.3730

    Single vesicle docking and fusion assay. ( a ) tSNARE vesicles (t-vesicles) were immobilized via biotin-neutravidin coupling onto a PEG-coated glass surface. Complexation of individual vSNARE vesicles (v-vesicles) and t-vesicles was followed by fluorescence
    Figure Legend Snippet: Single vesicle docking and fusion assay. ( a ) tSNARE vesicles (t-vesicles) were immobilized via biotin-neutravidin coupling onto a PEG-coated glass surface. Complexation of individual vSNARE vesicles (v-vesicles) and t-vesicles was followed by fluorescence

    Techniques Used: Single Vesicle Fusion Assay, Fluorescence

    6) Product Images from "Time-resolved microscopy for imaging lanthanide luminescence in living cells"

    Article Title: Time-resolved microscopy for imaging lanthanide luminescence in living cells

    Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

    doi: 10.1002/cyto.a.20964

    Spectra, luminescent lifetime, and photobleaching kinetics of europium microspheres used in this study. (a) Normalized excitation (dotted) and emission spectra (solid) of 40 nm, NeutrAvidin ® -labeled europium microspheres (Fluospheres ™
    Figure Legend Snippet: Spectra, luminescent lifetime, and photobleaching kinetics of europium microspheres used in this study. (a) Normalized excitation (dotted) and emission spectra (solid) of 40 nm, NeutrAvidin ® -labeled europium microspheres (Fluospheres ™

    Techniques Used: Labeling

    7) Product Images from "Diffusive tail anchorage determines velocity and force produced by kinesin-14 between crosslinked microtubules"

    Article Title: Diffusive tail anchorage determines velocity and force produced by kinesin-14 between crosslinked microtubules

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04656-0

    Diffusively anchored kinesin-14 motors exert sub-pN forces. a Sparsely cy5-labeled, digoxigeninated microtubules were surface immobilized using anti-digoxigenin antibodies. The surface was passivated using the polymer pluronic F127. Full-length Ncd (flNcd) motors crosslinked surface-immobilized microtubules and transported microtubules, which were densely cy5-labeled and biotinylated (sliding assay geometry). A NeutrAvidin-coated microsphere bound to the transported microtubule was manipulated using optical tweezers. b Velocity distribution of microtubules transported by flNcd ( N = 265). c . For the boxplot elements description see Methods. d Characteristic force trace for a microsphere attached to a microtubule that is transported by flNcd. Note that once the microsphere attaches to the microtubule, sliding motion stops immediately and the microsphere is not transported out of the trap center. Direction of microtubule sliding is parallel to the x- axis. The binding event is marked with a red arrow. The last movement of the microscope stage prior to binding is marked with a vertical red line. e Typical force-velocity measurement used to quantify the stopping force of flNcd in microtubule overlaps. Below, a schematic zoom-in is shown. f Stopping force distribution of 33 independent transported microtubules (for method of force quantification see Fig. 2e). g . h Schematic depiction of the microtubule buckling assay. Two sets of microtubules were surface immobilized using anti-digoxigenin antibodies: dimly rhodamine-labeled digoxigeninated and cy5-labeled, biotinylated, digoxigeninated microtubules. NeutrAvidin was used to coat the biotinylated microtubules. flNcd was added and biotinylated, brightly rhodamine-labeled transport microtubules were allowed to bind. Upon encountering NeutrAvidin the transported microtubule stopped and, if Ncd exerted sufficient force, buckled. i Micrographs showing the buckling event with the highest force, which was calculated to be 1.1 pN. Several tens of seconds of thermal fluctuation preceded the buckling; scale bar 5 µm
    Figure Legend Snippet: Diffusively anchored kinesin-14 motors exert sub-pN forces. a Sparsely cy5-labeled, digoxigeninated microtubules were surface immobilized using anti-digoxigenin antibodies. The surface was passivated using the polymer pluronic F127. Full-length Ncd (flNcd) motors crosslinked surface-immobilized microtubules and transported microtubules, which were densely cy5-labeled and biotinylated (sliding assay geometry). A NeutrAvidin-coated microsphere bound to the transported microtubule was manipulated using optical tweezers. b Velocity distribution of microtubules transported by flNcd ( N = 265). c . For the boxplot elements description see Methods. d Characteristic force trace for a microsphere attached to a microtubule that is transported by flNcd. Note that once the microsphere attaches to the microtubule, sliding motion stops immediately and the microsphere is not transported out of the trap center. Direction of microtubule sliding is parallel to the x- axis. The binding event is marked with a red arrow. The last movement of the microscope stage prior to binding is marked with a vertical red line. e Typical force-velocity measurement used to quantify the stopping force of flNcd in microtubule overlaps. Below, a schematic zoom-in is shown. f Stopping force distribution of 33 independent transported microtubules (for method of force quantification see Fig. 2e). g . h Schematic depiction of the microtubule buckling assay. Two sets of microtubules were surface immobilized using anti-digoxigenin antibodies: dimly rhodamine-labeled digoxigeninated and cy5-labeled, biotinylated, digoxigeninated microtubules. NeutrAvidin was used to coat the biotinylated microtubules. flNcd was added and biotinylated, brightly rhodamine-labeled transport microtubules were allowed to bind. Upon encountering NeutrAvidin the transported microtubule stopped and, if Ncd exerted sufficient force, buckled. i Micrographs showing the buckling event with the highest force, which was calculated to be 1.1 pN. Several tens of seconds of thermal fluctuation preceded the buckling; scale bar 5 µm

    Techniques Used: Labeling, Binding Assay, Microscopy

    8) Product Images from "A nanopore machine promotes the vectorial transport of DNA across membranes"

    Article Title: A nanopore machine promotes the vectorial transport of DNA across membranes

    Journal: Nature Communications

    doi: 10.1038/ncomms3415

    Formation of a nanopore–DNA rotaxane. ( a ) Representation of the hybridization of the DNA molecules used to form the rotaxane. Arrowheads mark the 3′-ends of strands. The suffix bio indicates a biotin moiety. ( b ) Rotaxane formation. At −100 mV following the addition of the DNA hybrid 3 (1.0 μM) complexed with neutravidin (0.3 μM, tetramer) and oligo 4 (1.0 μM) to the trans compartment, the open pore current of ClyA- 2 ( I O−100 =−1.71±0.07 nA, mean±s.d., n =4) was reduced to level 1 −100 =−1.1±0.04 nA (mean±s.d., I RES−100 value of 0.64±0.02, mean±s.d., n =4), indicating that dsDNA threaded the pore from the trans side. Stepping to +100 mV (red asterisk) produced a current block with I RES+100 =0.77±0.04 (mean±s.d., level 2 +100 =1.31±0.09 nA, mean±s.d., n =4), indicating that the DNA was still occupying the pore at positive applied potentials. Level 2 most likely corresponded to ssDNA 2 occupying the vestibule of the pore. Successive switching to positive and negative applied potentials did not restore the open pore current, confirming that a rotaxane was permanently formed. ( c – e ) Rotaxane removal. ( c ) At +100 mV, the ionic current through ClyA- 2 nanopores showed a multitude of fast current blockades (see also Supplementary Fig. S1 ), suggesting that the ssDNA molecules attached to the cis entrance transiently entered the lumen of the pore. ( d ) After the rotaxane was formed ( Fig. 2b ), at +100 mV the nanopore showed a steady ionic current (level 2 +100 ), suggesting that a single DNA molecule was occupying the pore. ( e ) Twenty minutes after the addition of 20 mM DTT to the cis compartment, the DNA molecules atop the ClyA pore were removed by disulphide bond exchange and the open pore current at +100 mV was restored ( I O+100 =1.78±0.07 nA, mean±s.d., n =4). The electrical recordings were carried out in 2.5 M NaCl, 15 mM Tris HCl, pH 7.5, at 22 °C. Data were recorded by applying a 10-kHz low-pass Bessel filter and using a 20 μs (50 kHz) sampling rate.
    Figure Legend Snippet: Formation of a nanopore–DNA rotaxane. ( a ) Representation of the hybridization of the DNA molecules used to form the rotaxane. Arrowheads mark the 3′-ends of strands. The suffix bio indicates a biotin moiety. ( b ) Rotaxane formation. At −100 mV following the addition of the DNA hybrid 3 (1.0 μM) complexed with neutravidin (0.3 μM, tetramer) and oligo 4 (1.0 μM) to the trans compartment, the open pore current of ClyA- 2 ( I O−100 =−1.71±0.07 nA, mean±s.d., n =4) was reduced to level 1 −100 =−1.1±0.04 nA (mean±s.d., I RES−100 value of 0.64±0.02, mean±s.d., n =4), indicating that dsDNA threaded the pore from the trans side. Stepping to +100 mV (red asterisk) produced a current block with I RES+100 =0.77±0.04 (mean±s.d., level 2 +100 =1.31±0.09 nA, mean±s.d., n =4), indicating that the DNA was still occupying the pore at positive applied potentials. Level 2 most likely corresponded to ssDNA 2 occupying the vestibule of the pore. Successive switching to positive and negative applied potentials did not restore the open pore current, confirming that a rotaxane was permanently formed. ( c – e ) Rotaxane removal. ( c ) At +100 mV, the ionic current through ClyA- 2 nanopores showed a multitude of fast current blockades (see also Supplementary Fig. S1 ), suggesting that the ssDNA molecules attached to the cis entrance transiently entered the lumen of the pore. ( d ) After the rotaxane was formed ( Fig. 2b ), at +100 mV the nanopore showed a steady ionic current (level 2 +100 ), suggesting that a single DNA molecule was occupying the pore. ( e ) Twenty minutes after the addition of 20 mM DTT to the cis compartment, the DNA molecules atop the ClyA pore were removed by disulphide bond exchange and the open pore current at +100 mV was restored ( I O+100 =1.78±0.07 nA, mean±s.d., n =4). The electrical recordings were carried out in 2.5 M NaCl, 15 mM Tris HCl, pH 7.5, at 22 °C. Data were recorded by applying a 10-kHz low-pass Bessel filter and using a 20 μs (50 kHz) sampling rate.

    Techniques Used: Hybridization, Produced, Blocking Assay, Sampling

    dsDNA translocation through ClyA nanopores. On the right of the current traces, ClyA and neutravidin are depicted as grey and red cartoons, respectively, and the dsDNA is shown as a black double line. ( a ) A section through ClyA from S. typhi constructed by homology modelling from the E. coli ClyA structure (PDB: 2WCD , 90% sequence identity) 25 . ClyA is shown as surface representation and coloured according to the ‘in vacuo’ electrostatics (red for negative regions, and blue for positive regions, Pymol). The arrow shows one of the 12 residues of 103 (serine in the WT sequence). The pore dimensions are given considering the van der Waals radii of the atoms. ( b ) At +100 mV, the addition of 0.12 μM of 290 bp dsDNA 1 to the cis side of a ClyA nanopore provoked short current blockades of I RES =0.63±0.01 (mean±s.d., level I O+100 =1.74±0.05 nA, mean±s.d. and level 1* +100 =1.10±0.03 nA, mean±s.d., n =3) that were because of the translocation of dsDNA through the pore. The addition of neutravidin to the cis chamber converted the short current blockades to higher conductance and long-lasting current blockades with I RES+100 =0.68±0.01 (mean±s.d., level 1 +100 =1.19±0.01 nA, mean±s.d., n =4) due to the threading of DNA through the pore. The open pore current was restored by reversing the potential to −100 mV (red asterisks) and the stepping back to +100 mV. The inset shows a typical transient current blockade. ( c ) Details of a current blockade because of the formation of a cis pseudorotaxane at +100 mV. ( d ) Formation of a trans pseudorotaxane at −100 mV by threading the dsDNA:neutravidin complex (see above) from the trans side (level 1 −100 =−1.02±0.03 nA, mean±s.d., I RES-100 =0.62±0.01, mean±s.d., n =4). The electrical recordings were carried out in 2.5 M NaCl, 15 mM Tris HCl, pH 7.5 at 22 °C. Data were recorded by applying a 10-kHz low-pass Bessel filter and using a 20μs (50 kHz) sampling rate.
    Figure Legend Snippet: dsDNA translocation through ClyA nanopores. On the right of the current traces, ClyA and neutravidin are depicted as grey and red cartoons, respectively, and the dsDNA is shown as a black double line. ( a ) A section through ClyA from S. typhi constructed by homology modelling from the E. coli ClyA structure (PDB: 2WCD , 90% sequence identity) 25 . ClyA is shown as surface representation and coloured according to the ‘in vacuo’ electrostatics (red for negative regions, and blue for positive regions, Pymol). The arrow shows one of the 12 residues of 103 (serine in the WT sequence). The pore dimensions are given considering the van der Waals radii of the atoms. ( b ) At +100 mV, the addition of 0.12 μM of 290 bp dsDNA 1 to the cis side of a ClyA nanopore provoked short current blockades of I RES =0.63±0.01 (mean±s.d., level I O+100 =1.74±0.05 nA, mean±s.d. and level 1* +100 =1.10±0.03 nA, mean±s.d., n =3) that were because of the translocation of dsDNA through the pore. The addition of neutravidin to the cis chamber converted the short current blockades to higher conductance and long-lasting current blockades with I RES+100 =0.68±0.01 (mean±s.d., level 1 +100 =1.19±0.01 nA, mean±s.d., n =4) due to the threading of DNA through the pore. The open pore current was restored by reversing the potential to −100 mV (red asterisks) and the stepping back to +100 mV. The inset shows a typical transient current blockade. ( c ) Details of a current blockade because of the formation of a cis pseudorotaxane at +100 mV. ( d ) Formation of a trans pseudorotaxane at −100 mV by threading the dsDNA:neutravidin complex (see above) from the trans side (level 1 −100 =−1.02±0.03 nA, mean±s.d., I RES-100 =0.62±0.01, mean±s.d., n =4). The electrical recordings were carried out in 2.5 M NaCl, 15 mM Tris HCl, pH 7.5 at 22 °C. Data were recorded by applying a 10-kHz low-pass Bessel filter and using a 20μs (50 kHz) sampling rate.

    Techniques Used: Translocation Assay, Construct, Sequencing, Sampling

    ssDNA and dsDNA blockades of ClyA-CS. At +100 mV, the addition of 2.0 μM of a biotinylated ssDNA ( 5a ) to the cis side of ClyA-CS nanopores in the presence of 0.6 μM neutravidin (tetramer) provoked transient current blockades (level 2* +100 =1.24±0.02 nA, mean±s.d., I RES+100 =0.69±0.04, mean±s.d., n =3), indicating that ssDNA entered the lumen of the pore but only transiently. The subsequent addition of 2.0 μM of the complementary ssDNA strand ( 5b ) to the cis compartment converted the current blockades into permanent level 1 +100 blocks (1.22±0.13 nA, mean±s.d., I RES+100 =0.67±0.01, mean±s.d., n =3), indicating that the dsDNA so formed spanned the entire length of the pore. After each DNA capture, the open pore was regenerated by manual reversal of the potential to −100 mV. Spikes above and below the open pore current level represent capacitive transients. The electrical recordings were carried out in 2.5 M NaCl, 15 mM Tris HCl, pH 7.5, at 22 °C. Data were recorded by applying a 10-kHz low-pass Bessel filter and using a 20μs (50 kHz) sampling rate.
    Figure Legend Snippet: ssDNA and dsDNA blockades of ClyA-CS. At +100 mV, the addition of 2.0 μM of a biotinylated ssDNA ( 5a ) to the cis side of ClyA-CS nanopores in the presence of 0.6 μM neutravidin (tetramer) provoked transient current blockades (level 2* +100 =1.24±0.02 nA, mean±s.d., I RES+100 =0.69±0.04, mean±s.d., n =3), indicating that ssDNA entered the lumen of the pore but only transiently. The subsequent addition of 2.0 μM of the complementary ssDNA strand ( 5b ) to the cis compartment converted the current blockades into permanent level 1 +100 blocks (1.22±0.13 nA, mean±s.d., I RES+100 =0.67±0.01, mean±s.d., n =3), indicating that the dsDNA so formed spanned the entire length of the pore. After each DNA capture, the open pore was regenerated by manual reversal of the potential to −100 mV. Spikes above and below the open pore current level represent capacitive transients. The electrical recordings were carried out in 2.5 M NaCl, 15 mM Tris HCl, pH 7.5, at 22 °C. Data were recorded by applying a 10-kHz low-pass Bessel filter and using a 20μs (50 kHz) sampling rate.

    Techniques Used: Sampling

    Transport of DNA through ClyA nanopores. ( a ) Schematic representation of the strand-displacement reaction that promoted the release of DNA from the pore. The suffix bio indicates a biotin moiety. ( b ) At +50 mV and in the presence of 3 (0.3 μM), ClyA- 2 showed a steady open pore current ( I O+50 =0.85±0.01 nA, mean±s.d., n =3), showing that the ssDNA strands attached to the pore did not thread through the lumen of the pore and prevented the translocation of dsDNA form solution. ( c ) The addition of the ssDNA strand 6 (40 nM) to the cis chamber in the presence of the strand 3 and 0.3 μM neutravidin (tetramer) in the trans compartment produced long-lasting current blockades with I RES+50 =0.70±0.02 (mean±s.d., level 2 +50 =0.59±0.02 nA, mean±s.d., n =5), indicating that the dsDNA hybrid threaded the pore. ( d ) The subsequent addition of 1 μM of 7 to the trans chamber (+50 mV) promoted the release of the DNA thread by a strand displacement reaction. 2 returned to the cis compartment against the applied potential and the open pore current was restored. Specific dsDNA molecules were then sequentially captured and released as shown by successive blocked and open pore currents. In this experiment, the applied potential was set to +50 mV and not to +100 mV as in the rest of this work. This is because at +100 mV ClyA- 2 occasionally produced long current blockades that were similar to typical events provoked by the capture of non-specific DNA ( Supplementary Fig. S5c ). Such current blockades were not observed at lower applied potentials; hence, we performed this experiment at +50 mV. In addition, at+50 mV the rate of DNA capture was lower than at +100 mV, and therefore the cycles of capture and release were more easily observed. The electrical recordings were carried out in 2.5 M NaCl, 15 mM Tris HCl, pH 7.5 at 22 °C. Data were recorded by applying a 2-kHz low-pass Bessel filter and using a 100μs (10 kHz) sampling rate.
    Figure Legend Snippet: Transport of DNA through ClyA nanopores. ( a ) Schematic representation of the strand-displacement reaction that promoted the release of DNA from the pore. The suffix bio indicates a biotin moiety. ( b ) At +50 mV and in the presence of 3 (0.3 μM), ClyA- 2 showed a steady open pore current ( I O+50 =0.85±0.01 nA, mean±s.d., n =3), showing that the ssDNA strands attached to the pore did not thread through the lumen of the pore and prevented the translocation of dsDNA form solution. ( c ) The addition of the ssDNA strand 6 (40 nM) to the cis chamber in the presence of the strand 3 and 0.3 μM neutravidin (tetramer) in the trans compartment produced long-lasting current blockades with I RES+50 =0.70±0.02 (mean±s.d., level 2 +50 =0.59±0.02 nA, mean±s.d., n =5), indicating that the dsDNA hybrid threaded the pore. ( d ) The subsequent addition of 1 μM of 7 to the trans chamber (+50 mV) promoted the release of the DNA thread by a strand displacement reaction. 2 returned to the cis compartment against the applied potential and the open pore current was restored. Specific dsDNA molecules were then sequentially captured and released as shown by successive blocked and open pore currents. In this experiment, the applied potential was set to +50 mV and not to +100 mV as in the rest of this work. This is because at +100 mV ClyA- 2 occasionally produced long current blockades that were similar to typical events provoked by the capture of non-specific DNA ( Supplementary Fig. S5c ). Such current blockades were not observed at lower applied potentials; hence, we performed this experiment at +50 mV. In addition, at+50 mV the rate of DNA capture was lower than at +100 mV, and therefore the cycles of capture and release were more easily observed. The electrical recordings were carried out in 2.5 M NaCl, 15 mM Tris HCl, pH 7.5 at 22 °C. Data were recorded by applying a 2-kHz low-pass Bessel filter and using a 100μs (10 kHz) sampling rate.

    Techniques Used: Translocation Assay, Produced, Sampling

    9) Product Images from "Dynamics of human telomerase recruitment depend on template-telomere base pairing"

    Article Title: Dynamics of human telomerase recruitment depend on template-telomere base pairing

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E17-11-0637

    Imetelstat (GRN163L) is a competitive inhibitor of primer-substrate binding by telomerase. (A) Experimental design of single-molecule telomerase primer binding and activity assay. Halo-telomerase is modified with a biotin-HaloTag-ligand and immobilized on the coverslip surface using NeutrAvidin. Primer binding is visualized by telomerase-dependent recruitment of a fluorescent primer to the coverslip surface. The telomerase extension product is detected using a fluorescently labeled oligonucleotide anti-sense to the telomerase extension product. (B) Western blot and fluorescence imaging of Halo-telomerase modified with a fluorescent dye (JF646) or biotin, probed with an anti-TERT antibody or HRP-conjugated streptavidin. (C) Single-molecule TIRF imaging of primer molecules recruited to the coverslip surface by telomerase (top) and its colocalization with telomerase extension products after incubation with nucleotide substrate (bottom). (D) Single-molecule TIRF imaging of primer binding by telomerase in the presence of increasing concentrations of imetelstat. (E) Quantification of primer binding to telomerase as a function of imetelstat concentration ( n = 5 fields of view per concentration, data points plotted as mean ± SD, error on IC 50 reflects error in the corresponding fit of the data to a simple binding curve). (F) Direct telomerase assay at 150 mM KCl in the absence and presence of imetelstat (10 nM), or mismatched control oligonucleotide (MM Control, 10 nM), and increasing concentrations of primer substrate. LC1, LC2, and LC3, labeled DNA loading controls. (G) Quantification of telomerase activity as a function of primer concentration in absence and presence of imetelstat (10 nM) or mismatched control oligonucleotide (MM Control, 10 nM).
    Figure Legend Snippet: Imetelstat (GRN163L) is a competitive inhibitor of primer-substrate binding by telomerase. (A) Experimental design of single-molecule telomerase primer binding and activity assay. Halo-telomerase is modified with a biotin-HaloTag-ligand and immobilized on the coverslip surface using NeutrAvidin. Primer binding is visualized by telomerase-dependent recruitment of a fluorescent primer to the coverslip surface. The telomerase extension product is detected using a fluorescently labeled oligonucleotide anti-sense to the telomerase extension product. (B) Western blot and fluorescence imaging of Halo-telomerase modified with a fluorescent dye (JF646) or biotin, probed with an anti-TERT antibody or HRP-conjugated streptavidin. (C) Single-molecule TIRF imaging of primer molecules recruited to the coverslip surface by telomerase (top) and its colocalization with telomerase extension products after incubation with nucleotide substrate (bottom). (D) Single-molecule TIRF imaging of primer binding by telomerase in the presence of increasing concentrations of imetelstat. (E) Quantification of primer binding to telomerase as a function of imetelstat concentration ( n = 5 fields of view per concentration, data points plotted as mean ± SD, error on IC 50 reflects error in the corresponding fit of the data to a simple binding curve). (F) Direct telomerase assay at 150 mM KCl in the absence and presence of imetelstat (10 nM), or mismatched control oligonucleotide (MM Control, 10 nM), and increasing concentrations of primer substrate. LC1, LC2, and LC3, labeled DNA loading controls. (G) Quantification of telomerase activity as a function of primer concentration in absence and presence of imetelstat (10 nM) or mismatched control oligonucleotide (MM Control, 10 nM).

    Techniques Used: Binding Assay, Activity Assay, Modification, Labeling, Western Blot, Fluorescence, Imaging, Incubation, Concentration Assay, Telomerase Assay

    10) Product Images from "Pharmacophore Generation from a Drug-like Core Molecule Surrounded by a Library Peptide via the 10BASEd-T on Bacteriophage T7"

    Article Title: Pharmacophore Generation from a Drug-like Core Molecule Surrounded by a Library Peptide via the 10BASEd-T on Bacteriophage T7

    Journal: Molecules

    doi: 10.3390/molecules19022481

    ( A ) Determination of streptavidin-binding affinities of the Sal-conjugated peptide (left panel) and the mock peptide (right panel) by fluorescence polarization assay. The amino acid sequence of the peptide is shown in the upper part. The plots indicate the polarization (mP) of the fluorophore (FAM)-coupled peptides in the presence of various concentrations of target proteins. NeutrAvidin and bovine serum albumin (BSA) were used as mock target proteins. ( B ) Competitive binding assay. Biotin as a competitor was mixed with the Sal-conjugated peptide-streptavidin complex. Error bars represent standard deviations.
    Figure Legend Snippet: ( A ) Determination of streptavidin-binding affinities of the Sal-conjugated peptide (left panel) and the mock peptide (right panel) by fluorescence polarization assay. The amino acid sequence of the peptide is shown in the upper part. The plots indicate the polarization (mP) of the fluorophore (FAM)-coupled peptides in the presence of various concentrations of target proteins. NeutrAvidin and bovine serum albumin (BSA) were used as mock target proteins. ( B ) Competitive binding assay. Biotin as a competitor was mixed with the Sal-conjugated peptide-streptavidin complex. Error bars represent standard deviations.

    Techniques Used: Binding Assay, Fluorescence, Sequencing, Competitive Binding Assay

    11) Product Images from "Cooperative Nucleotide Binding in Hsp90 and Its Regulation by Aha1"

    Article Title: Cooperative Nucleotide Binding in Hsp90 and Its Regulation by Aha1

    Journal: Biophysical Journal

    doi: 10.1016/j.bpj.2017.08.032

    Data acquisition, data analysis, and state allocation. ( a ) Pictogram of the studied system consisting of an Hsp90 dimer with the labels Atto488 ( blue ) and Atto550 ( green ) attached and the reporter nucleotide AMP-PNP ∗ in solution, labeled with Atto647N ( red ). The protein is immobilized by NeutrAvidin/biotin interaction on the top of the flow chamber and excited by an evanescent field using alternating laser excitation with a blue and a green laser in a prism-type total internal reflection fluorescence geometry. The fluorescence light is collected by an objective, separated by dichroic mirrors, and detected with electron-multiplying charge-coupled device cameras. ( b ) Fluorescence intensity traces of a single particle after the excitation of the blue ( top ) and the green ( center ) dye measured in the absence of additional unlabeled nucleotide or co-chaperone. The partial fluorescence traces ( P F em ex , with excitation of the ex dye and emission of the em dye) calculated from the intensity traces are shown below. ( c ) Pictograms of the distinguishable states and the respective identifiers used in this work. The first two populations represent the same functional state, namely open Hsp90 with nucleotide bound. ( d ) 3D representation of the Gaussians (isosurface at FWHM) fitted to the partial fluorescence data, which represent the five different populations. The same color code as in ( c ) is used. ( e ) The resulting state allocation for the fluorescence intensity traces is shown in ( b ).
    Figure Legend Snippet: Data acquisition, data analysis, and state allocation. ( a ) Pictogram of the studied system consisting of an Hsp90 dimer with the labels Atto488 ( blue ) and Atto550 ( green ) attached and the reporter nucleotide AMP-PNP ∗ in solution, labeled with Atto647N ( red ). The protein is immobilized by NeutrAvidin/biotin interaction on the top of the flow chamber and excited by an evanescent field using alternating laser excitation with a blue and a green laser in a prism-type total internal reflection fluorescence geometry. The fluorescence light is collected by an objective, separated by dichroic mirrors, and detected with electron-multiplying charge-coupled device cameras. ( b ) Fluorescence intensity traces of a single particle after the excitation of the blue ( top ) and the green ( center ) dye measured in the absence of additional unlabeled nucleotide or co-chaperone. The partial fluorescence traces ( P F em ex , with excitation of the ex dye and emission of the em dye) calculated from the intensity traces are shown below. ( c ) Pictograms of the distinguishable states and the respective identifiers used in this work. The first two populations represent the same functional state, namely open Hsp90 with nucleotide bound. ( d ) 3D representation of the Gaussians (isosurface at FWHM) fitted to the partial fluorescence data, which represent the five different populations. The same color code as in ( c ) is used. ( e ) The resulting state allocation for the fluorescence intensity traces is shown in ( b ).

    Techniques Used: Labeling, Flow Cytometry, Fluorescence, Functional Assay

    12) Product Images from "Viral suppressors of RNAi employ a rapid screening mode to discriminate viral RNA from cellular small RNA"

    Article Title: Viral suppressors of RNAi employ a rapid screening mode to discriminate viral RNA from cellular small RNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx1316

    Development of a single-molecule assay for real-time observation of viral RNAs recognition by Dcr-2 complex. ( A ) Schematic representation of sample preparation. Dcr-2 was constructed with 6xHis, TEV and AP tags which were used for Ni-NTA purification, elution, and in vivo biotinylation, respectively. Dcr-2 proteins were biotinylated in Sf9 cells. The protein was purified using Ni-NTA column and eluted by 6xHis-tag cleavage by the TEV protease. ( B ) Western blot analysis displays the efficiency of in vivo biotinylation of AP-tagged Dcr-2 in the presence (lane 1) or absence (lane 2) of 1 mg/mL free biotin in the culture medium. The biotinylated Dcr-2 bound to StreptAvidin, which resulted in the shift observed in lane 1. ( C ) In vitro cleavage assay of Cy5-labeled 70-nt dsRNA with blunt end by Dcr-2–LqPD in the absence (lane 1) and presence (lane 2) of 10 mM MgCl 2 . The top band indicates non-cleaved dsRNA, and the lower bands indicate cleavage products. ( D ) Schematic representation of single-molecule immobilization. Dcr-2 was conjugated to a polymer-coated surface via NeutrAvidin-biotin interaction. Contaminant proteins were washed away before the introduction of LqPD recombinant protein into the imaging chamber. Dcr-2 and LqPD were incubated together for 5 min to promote protein–protein interaction on the surface of the imaging chamber. Non-bound LqPD was washed away before Cy5-labeled 70-nt dsRNA was introduced. Interactions between the surface-immobilized Dcr-2 complexes with Cy5-labeled dsRNA were visualized through TIRF microscopy. Dots in the EM-CCD image reflect docking of dsRNA to individual Dcr-2–LqPD complexes. The EM-CCD image illustrated the binding events over 25 × 25 μm field of view. Scale bar, 5 μm. ( E ) EM-CCD images illustrating stable docking of Cy5-labeled dsRNA to passivated surface without any protein (left), passivated surface with LqPD non-specifically immobilized (second image), surface immobilized Dcr-2 in the absence of LqPD (third image) and surface immobilized Dcr-2–LqPD complexes. Scale bar, 5 μm. The histogram (right panel) compares the absolute binding activity of Dcr-2 alone and Dcr-2–LqPD complex. Data are presented as averages and SD of three independent experiments. In each experiment snapshots from 10 fields of view were analyzed. ( F ) Representative time traces (at a time resolution of 300 ms) exhibiting recognition of multiple Cy5-labeled 70-nt dsRNA by a single Dcr-2–LqPD complex. The dwell-time (Δ τ ) is the time between docking and dissociation. The 70-nt Cy5-dsRNA was added at a time period of 5 s. ( G ) Dwell-time histogram derived from binding events recorded for 450 s in a pre-steady state condition. The distribution was fitted with a single exponential decay (gray line) where the average dwell-time is Δ τ = 12.5 ± 2.2 s. Data are presented as average and SD of three independent experiments.
    Figure Legend Snippet: Development of a single-molecule assay for real-time observation of viral RNAs recognition by Dcr-2 complex. ( A ) Schematic representation of sample preparation. Dcr-2 was constructed with 6xHis, TEV and AP tags which were used for Ni-NTA purification, elution, and in vivo biotinylation, respectively. Dcr-2 proteins were biotinylated in Sf9 cells. The protein was purified using Ni-NTA column and eluted by 6xHis-tag cleavage by the TEV protease. ( B ) Western blot analysis displays the efficiency of in vivo biotinylation of AP-tagged Dcr-2 in the presence (lane 1) or absence (lane 2) of 1 mg/mL free biotin in the culture medium. The biotinylated Dcr-2 bound to StreptAvidin, which resulted in the shift observed in lane 1. ( C ) In vitro cleavage assay of Cy5-labeled 70-nt dsRNA with blunt end by Dcr-2–LqPD in the absence (lane 1) and presence (lane 2) of 10 mM MgCl 2 . The top band indicates non-cleaved dsRNA, and the lower bands indicate cleavage products. ( D ) Schematic representation of single-molecule immobilization. Dcr-2 was conjugated to a polymer-coated surface via NeutrAvidin-biotin interaction. Contaminant proteins were washed away before the introduction of LqPD recombinant protein into the imaging chamber. Dcr-2 and LqPD were incubated together for 5 min to promote protein–protein interaction on the surface of the imaging chamber. Non-bound LqPD was washed away before Cy5-labeled 70-nt dsRNA was introduced. Interactions between the surface-immobilized Dcr-2 complexes with Cy5-labeled dsRNA were visualized through TIRF microscopy. Dots in the EM-CCD image reflect docking of dsRNA to individual Dcr-2–LqPD complexes. The EM-CCD image illustrated the binding events over 25 × 25 μm field of view. Scale bar, 5 μm. ( E ) EM-CCD images illustrating stable docking of Cy5-labeled dsRNA to passivated surface without any protein (left), passivated surface with LqPD non-specifically immobilized (second image), surface immobilized Dcr-2 in the absence of LqPD (third image) and surface immobilized Dcr-2–LqPD complexes. Scale bar, 5 μm. The histogram (right panel) compares the absolute binding activity of Dcr-2 alone and Dcr-2–LqPD complex. Data are presented as averages and SD of three independent experiments. In each experiment snapshots from 10 fields of view were analyzed. ( F ) Representative time traces (at a time resolution of 300 ms) exhibiting recognition of multiple Cy5-labeled 70-nt dsRNA by a single Dcr-2–LqPD complex. The dwell-time (Δ τ ) is the time between docking and dissociation. The 70-nt Cy5-dsRNA was added at a time period of 5 s. ( G ) Dwell-time histogram derived from binding events recorded for 450 s in a pre-steady state condition. The distribution was fitted with a single exponential decay (gray line) where the average dwell-time is Δ τ = 12.5 ± 2.2 s. Data are presented as average and SD of three independent experiments.

    Techniques Used: Sample Prep, Construct, Purification, In Vivo, Western Blot, In Vitro, Cleavage Assay, Labeling, Recombinant, Imaging, Incubation, Microscopy, Binding Assay, Activity Assay, Mass Spectrometry, Derivative Assay

    VSRs stably bind to long dsRNA molecules. ( A ) Schematic representation of single-molecule assay to visualize dsRNA recognition by VSRs in steady state conditions. Biotinylated Anti-MBP antibody was conjugated to a polymer-coated surface via NeutrAvidin–biotin interaction. VSRs were incubated for 5 min with the surface-immobilized anti-MBP to promote the interaction on the surface of the imaging chamber. Non-bound VSRs were washed away before Cy5-labeled 70-nt dsRNA was introduced. VSRs and dsRNA were incubated in the imaging for 5 min to reach equilibrium. The unbound dsRNA molecules were washed away before imaging. ( B ) EM-CCD images illustrating dsRNA binding activity of surface-immobilized (50 nM) CYV-VP3, DXV-VP3 and DCV-1A at steady state conditions. Scale bar, 5 μm. ( C ) Quantification of dsRNA binding activity of surface-immobilized CYV-VP3, DXV-VP3 and DCV-1A. Data are normalized and presented as average and SD of three independent experiments. Snapshots from 10 fields of view were analyzed in each experiment. ( D, F and H ) Representative time traces (at a time resolution of 300 ms) reflecting the recognition of 70-nt dsRNA by CYV-VP3 (D), DXV-VP3 (F) and DCV-1A (H) in pre-steady state conditions. The Cy5-labeled 70-nt dsRNA was introduced in the imaging chamber at t = 5 s. ( E, G and I ) Dwell-time histogram derived from binding events to surface-immobilized CYV-VP3 (E), DXV-VP3 (G) and DCV-1A (I) recorded for 450 s in pre-steady state conditions. The distribution was fitted with a single exponential decay (gray line in E) or double exponential decay (gray line in G and I). Data are presented as average and SD of three independent experiments. The last bin at the end of the histograms represents the binding events that survived beyond 450 s of imagining. The pie charts (right panels) illustrate the percentage of short (red) and long (black) binding events. The cut-off between short and long binding is 10 s.
    Figure Legend Snippet: VSRs stably bind to long dsRNA molecules. ( A ) Schematic representation of single-molecule assay to visualize dsRNA recognition by VSRs in steady state conditions. Biotinylated Anti-MBP antibody was conjugated to a polymer-coated surface via NeutrAvidin–biotin interaction. VSRs were incubated for 5 min with the surface-immobilized anti-MBP to promote the interaction on the surface of the imaging chamber. Non-bound VSRs were washed away before Cy5-labeled 70-nt dsRNA was introduced. VSRs and dsRNA were incubated in the imaging for 5 min to reach equilibrium. The unbound dsRNA molecules were washed away before imaging. ( B ) EM-CCD images illustrating dsRNA binding activity of surface-immobilized (50 nM) CYV-VP3, DXV-VP3 and DCV-1A at steady state conditions. Scale bar, 5 μm. ( C ) Quantification of dsRNA binding activity of surface-immobilized CYV-VP3, DXV-VP3 and DCV-1A. Data are normalized and presented as average and SD of three independent experiments. Snapshots from 10 fields of view were analyzed in each experiment. ( D, F and H ) Representative time traces (at a time resolution of 300 ms) reflecting the recognition of 70-nt dsRNA by CYV-VP3 (D), DXV-VP3 (F) and DCV-1A (H) in pre-steady state conditions. The Cy5-labeled 70-nt dsRNA was introduced in the imaging chamber at t = 5 s. ( E, G and I ) Dwell-time histogram derived from binding events to surface-immobilized CYV-VP3 (E), DXV-VP3 (G) and DCV-1A (I) recorded for 450 s in pre-steady state conditions. The distribution was fitted with a single exponential decay (gray line in E) or double exponential decay (gray line in G and I). Data are presented as average and SD of three independent experiments. The last bin at the end of the histograms represents the binding events that survived beyond 450 s of imagining. The pie charts (right panels) illustrate the percentage of short (red) and long (black) binding events. The cut-off between short and long binding is 10 s.

    Techniques Used: Stable Transfection, Incubation, Imaging, Labeling, Binding Assay, Activity Assay, Mass Spectrometry, Derivative Assay

    13) Product Images from "DNA target sequence identification mechanism for dimer-active protein complexes"

    Article Title: DNA target sequence identification mechanism for dimer-active protein complexes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks1345

    TelK monomers diffuse along non-target DNA, whereas dimers immobilize. ( a ) Schematic of TIRFM experimental set-up and representative fluorescence image. An etched glass slide with 1 × 1 µm pedestals separated by 7-µm etches was coated with neutravidin. Dual-biotinylated λ-DNA was flowed in to form DNA bridges, and the chamber was, subsequently, incubated with QD-labelled TelK monomers (here, 340 nM). QDs stuck to the glass surface were used as reference spots to ensure that drift and background motion were minimal. TelK concentrations used in all TIRF experiments ranged from 70 to 1350 nM. ( b ) Kymograph of QD-labelled TelK on λ-DNA showing mobile (1–31 s) and stationary (32–50 s) states after analysis and background subtraction with Gaussian fitting of spots. ( c ) Fluorescence intensity corresponding to the kymograph. The intensity of the mobile TelK doubles as it becomes immobile along the λ-DNA bridge. QD-blinking events (arrows), known to occur for single QDs, are observed only before TelK immobilization. ( d ) Diffusion coefficient and lifetime for blinking fluorescence spots ( n = 114, red) and non-blinking spots ( n = 81, blue). The shaded area represents the limit of sensitivity of our assay. The distribution of diffusion coefficients (right panel) is bimodal, with blinkers diffusing approximately four orders of magnitude faster than non-blinkers. As shown in the lifetime distributions (bottom panel), blinking spots also remained DNA-bound for shorter times (4.2 s) than non-blinkers ( > 50 s).
    Figure Legend Snippet: TelK monomers diffuse along non-target DNA, whereas dimers immobilize. ( a ) Schematic of TIRFM experimental set-up and representative fluorescence image. An etched glass slide with 1 × 1 µm pedestals separated by 7-µm etches was coated with neutravidin. Dual-biotinylated λ-DNA was flowed in to form DNA bridges, and the chamber was, subsequently, incubated with QD-labelled TelK monomers (here, 340 nM). QDs stuck to the glass surface were used as reference spots to ensure that drift and background motion were minimal. TelK concentrations used in all TIRF experiments ranged from 70 to 1350 nM. ( b ) Kymograph of QD-labelled TelK on λ-DNA showing mobile (1–31 s) and stationary (32–50 s) states after analysis and background subtraction with Gaussian fitting of spots. ( c ) Fluorescence intensity corresponding to the kymograph. The intensity of the mobile TelK doubles as it becomes immobile along the λ-DNA bridge. QD-blinking events (arrows), known to occur for single QDs, are observed only before TelK immobilization. ( d ) Diffusion coefficient and lifetime for blinking fluorescence spots ( n = 114, red) and non-blinking spots ( n = 81, blue). The shaded area represents the limit of sensitivity of our assay. The distribution of diffusion coefficients (right panel) is bimodal, with blinkers diffusing approximately four orders of magnitude faster than non-blinkers. As shown in the lifetime distributions (bottom panel), blinking spots also remained DNA-bound for shorter times (4.2 s) than non-blinkers ( > 50 s).

    Techniques Used: Fluorescence, Incubation, Diffusion-based Assay

    14) Product Images from "Sub-cellular trafficking and functionality of 2?-O-methyl and 2?-O-methyl-phosphorothioate molecular beacons"

    Article Title: Sub-cellular trafficking and functionality of 2?-O-methyl and 2?-O-methyl-phosphorothioate molecular beacons

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp837

    Fluorescence images of MBs and MB-NeutrAvidin conjugates following microinjection in living MEF/3T3 cells. The representative images shown were acquired 20 min following the injection of ( A ) 2Me MBs, ( B ) 2Me MB-NeutrAvidin conjugates, ( C ) 2MePS MBs and ( D ), 2MePS MB-NeutrAvidin conjugates. The MBs used were not complementary to any known endogenous RNA in MEF/3T3 cells.
    Figure Legend Snippet: Fluorescence images of MBs and MB-NeutrAvidin conjugates following microinjection in living MEF/3T3 cells. The representative images shown were acquired 20 min following the injection of ( A ) 2Me MBs, ( B ) 2Me MB-NeutrAvidin conjugates, ( C ) 2MePS MBs and ( D ), 2MePS MB-NeutrAvidin conjugates. The MBs used were not complementary to any known endogenous RNA in MEF/3T3 cells.

    Techniques Used: Fluorescence, Injection

    Temporal measurements of non-specific MB opening, following microporation into MEF/3T3 cells. Nonsense ( A ) 2Me MBs (filled diamonds) and 2Me MB-NeutrAvidin conjugates (open circles) as well as ( B ) 2MePS MBs (filled diamonds) and 2MePS MB-NeutrAvidin conjugates (open circles) were microporated into living cells and fluorescence images were acquired over the course of 24 h. Quantification of MB opening was calculated as described in the ‘Materials and Methods’ section. Each data point represents that mean and standard deviation from at least 10 cells.
    Figure Legend Snippet: Temporal measurements of non-specific MB opening, following microporation into MEF/3T3 cells. Nonsense ( A ) 2Me MBs (filled diamonds) and 2Me MB-NeutrAvidin conjugates (open circles) as well as ( B ) 2MePS MBs (filled diamonds) and 2MePS MB-NeutrAvidin conjugates (open circles) were microporated into living cells and fluorescence images were acquired over the course of 24 h. Quantification of MB opening was calculated as described in the ‘Materials and Methods’ section. Each data point represents that mean and standard deviation from at least 10 cells.

    Techniques Used: Fluorescence, Standard Deviation

    Evaluation of MB functionality in MEF/3T3 cells at various times after microporation. Excess complementary nucleic acid targets were injected into MEF/3T3 cells 3 h and 24 h after being microporated with ( A ) 2Me MBs, ( B ) 2Me MB-NeutrAvidin conjugates, ( C ) 2MePS MBs and ( D ) 2MePS MB-NeutrAvidin conjugates. Fluorescent images of the cells were acquired immediately before and shortly after microinjection. Representative fluorescent images are shown.
    Figure Legend Snippet: Evaluation of MB functionality in MEF/3T3 cells at various times after microporation. Excess complementary nucleic acid targets were injected into MEF/3T3 cells 3 h and 24 h after being microporated with ( A ) 2Me MBs, ( B ) 2Me MB-NeutrAvidin conjugates, ( C ) 2MePS MBs and ( D ) 2MePS MB-NeutrAvidin conjugates. Fluorescent images of the cells were acquired immediately before and shortly after microinjection. Representative fluorescent images are shown.

    Techniques Used: Injection

    Fluorescence images of MBs and MB-NeutrAvidin conjugates in living MEF/3T3 cells at various time points after microporation. Images of the MB signals are shown for 2Me MBs at ( A ) 10 min, ( B ) 5 h and ( C ) 24 h and for 2Me MB-NeutrAvidin conjugates at ( D ) 10 min, ( E ) 5 h and ( F ) 24 h following microporation. Likewise, images of the 2MePS MBs acquired at ( G ) 10 min, ( H ), 5 h, ( I ) 24 h and images of the 2MePS MB-NeutrAvidin conjugates acquired at ( J ) 10 min, ( K ) 5 h, ( L ) or 24 h after microporation in MEF/3T3 cells are also shown.
    Figure Legend Snippet: Fluorescence images of MBs and MB-NeutrAvidin conjugates in living MEF/3T3 cells at various time points after microporation. Images of the MB signals are shown for 2Me MBs at ( A ) 10 min, ( B ) 5 h and ( C ) 24 h and for 2Me MB-NeutrAvidin conjugates at ( D ) 10 min, ( E ) 5 h and ( F ) 24 h following microporation. Likewise, images of the 2MePS MBs acquired at ( G ) 10 min, ( H ), 5 h, ( I ) 24 h and images of the 2MePS MB-NeutrAvidin conjugates acquired at ( J ) 10 min, ( K ) 5 h, ( L ) or 24 h after microporation in MEF/3T3 cells are also shown.

    Techniques Used: Fluorescence

    Temporal measurements of nonspecific MB opening, following microinjection into MEF/3T3 cells. Nonsense ( A ) 2Me MBs (filled diamonds) and 2Me MB-NeutrAvidin conjugates (open circles) as well as ( B ) 2MePS MBs (filled diamonds) and 2MePS MB-NeutrAvidin conjugates (open circles) were microinjected into living cells and fluorescence images were acquired every 5 min for 60 min. Quantification of MB opening was calculated as described in the ‘Materials and methods’ section. Each data point represents that mean and standard deviation from three to four cells.
    Figure Legend Snippet: Temporal measurements of nonspecific MB opening, following microinjection into MEF/3T3 cells. Nonsense ( A ) 2Me MBs (filled diamonds) and 2Me MB-NeutrAvidin conjugates (open circles) as well as ( B ) 2MePS MBs (filled diamonds) and 2MePS MB-NeutrAvidin conjugates (open circles) were microinjected into living cells and fluorescence images were acquired every 5 min for 60 min. Quantification of MB opening was calculated as described in the ‘Materials and methods’ section. Each data point represents that mean and standard deviation from three to four cells.

    Techniques Used: Fluorescence, Standard Deviation

    15) Product Images from "Charge, Diffusion, and Current Fluctuations of Single-Stranded DNA Trapped in an MspA Nanopore"

    Article Title: Charge, Diffusion, and Current Fluctuations of Single-Stranded DNA Trapped in an MspA Nanopore

    Journal: Biophysical Journal

    doi: 10.1016/j.bpj.2016.12.007

    ( A ) Schematic diagram of the experimental setup. A cutaway of the MspA protein is shown in green, while the ssDNA is red and the attached NeutrAvidin is gray. ( Light gray ) Lipid membrane, across which a voltage bias is applied. ( B ) After capture at 160 mV, each oligonucleotide exhibits a characteristic current blockage, shown in this composite trace from separate experiments. Data shown is the raw data acquired with a 10 kHz filter. ( C ) After capture at 160 mV, the NeutrAvidin is, to a great extent, butted up against the pore by the tension in the ssDNA, as shown in the top image. At lower voltages, diffusion leads to position fluctuations and eventual diffusive escape from the pore constriction ( top right ). Below the schematic is a typical voltage and current recording taken during an escape experiment on a single poly(dT) molecule. The time interval from the moment the voltage is reduced after a capture to the time when the DNA escapes from the pore constriction is the escape time for the event.
    Figure Legend Snippet: ( A ) Schematic diagram of the experimental setup. A cutaway of the MspA protein is shown in green, while the ssDNA is red and the attached NeutrAvidin is gray. ( Light gray ) Lipid membrane, across which a voltage bias is applied. ( B ) After capture at 160 mV, each oligonucleotide exhibits a characteristic current blockage, shown in this composite trace from separate experiments. Data shown is the raw data acquired with a 10 kHz filter. ( C ) After capture at 160 mV, the NeutrAvidin is, to a great extent, butted up against the pore by the tension in the ssDNA, as shown in the top image. At lower voltages, diffusion leads to position fluctuations and eventual diffusive escape from the pore constriction ( top right ). Below the schematic is a typical voltage and current recording taken during an escape experiment on a single poly(dT) molecule. The time interval from the moment the voltage is reduced after a capture to the time when the DNA escapes from the pore constriction is the escape time for the event.

    Techniques Used: Diffusion-based Assay

    16) Product Images from "Surface Presentation of Bioactive Ligands in a Non-Adhesive Background using DOPA-Tethered Biotinylated Poly(Ethylene Glycol)"

    Article Title: Surface Presentation of Bioactive Ligands in a Non-Adhesive Background using DOPA-Tethered Biotinylated Poly(Ethylene Glycol)

    Journal: Langmuir : the ACS journal of surfaces and colloids

    doi: 10.1021/la701415z

    Sequential adsorption of NeutrAvidin and various singly-biotinylated molecules to DPB/DP-coated surfaces was monitored using OWLS. (A) The surface concentrations of NeutrAvidin (solid circles), biotin-PEG600-cyclic LDV (cLDV; open circles), biotin-PEG5000
    Figure Legend Snippet: Sequential adsorption of NeutrAvidin and various singly-biotinylated molecules to DPB/DP-coated surfaces was monitored using OWLS. (A) The surface concentrations of NeutrAvidin (solid circles), biotin-PEG600-cyclic LDV (cLDV; open circles), biotin-PEG5000

    Techniques Used: Adsorption

    M07e cell interactions with immobilized biotin-SCF. (A) M07e cells adhered to surfaces prepared via sequential adsorption of NeutrAvidin and biotin-SCF in a DPB-dose-dependent manner. (B) Growth-factor-starved M07e cells incubated for 20 min on surfaces
    Figure Legend Snippet: M07e cell interactions with immobilized biotin-SCF. (A) M07e cells adhered to surfaces prepared via sequential adsorption of NeutrAvidin and biotin-SCF in a DPB-dose-dependent manner. (B) Growth-factor-starved M07e cells incubated for 20 min on surfaces

    Techniques Used: Adsorption, Incubation

    Adsorption of biotin-PEG600-cyclic RGD, alone and as a complex with NeutrAvidin, onto surfaces treated with DPB10. (A) Biotin-PEG600-cyclic RGD (cRGD) nonspecifically adsorbs to surfaces modified with DPB10 in the absence of NeutrAvidin. (B) In order
    Figure Legend Snippet: Adsorption of biotin-PEG600-cyclic RGD, alone and as a complex with NeutrAvidin, onto surfaces treated with DPB10. (A) Biotin-PEG600-cyclic RGD (cRGD) nonspecifically adsorbs to surfaces modified with DPB10 in the absence of NeutrAvidin. (B) In order

    Techniques Used: Adsorption, Modification

    M07e cell adhesion to biotinylated peptides. M07e cell adhesion onto DPB/DP surfaces prepared via sequential (open circles) and complex (solid circles) adsorption of NeutrAvidin and (A) biotin-PEG600-cyclic LDV or (B) biotin-PEG600-cyclic RGD. Complex
    Figure Legend Snippet: M07e cell adhesion to biotinylated peptides. M07e cell adhesion onto DPB/DP surfaces prepared via sequential (open circles) and complex (solid circles) adsorption of NeutrAvidin and (A) biotin-PEG600-cyclic LDV or (B) biotin-PEG600-cyclic RGD. Complex

    Techniques Used: Adsorption

    17) Product Images from "In vitro eradication of citrullinated protein specific B-lymphocytes of rheumatoid arthritis patients by targeted bifunctional nanoparticles"

    Article Title: In vitro eradication of citrullinated protein specific B-lymphocytes of rheumatoid arthritis patients by targeted bifunctional nanoparticles

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-016-0918-0

    Recognition of Cit-containing peptide epitope of fibrin β chain by antibodies in sera of RA patients and healthy blood donors a , b and by isolated B cells c . a Reactivities of RA ( n = 170) or healthy ( n = 138) serum samples with N-terminally bitotinylated β60-74Cit vs. β60-74Arg bound to neutravidin precoated plates. ELISA ratios were calculated (OD with β60-74Cit /OD with β60-74Arg). Data were analyzed with the Mann–Whitney test, and the median OD ratio of RA samples was 1.68, interquartile range 0.95–6.33, and the median OD ratio of healthy samples was 0.91, interquartile range 0.81–1.07 (*** p
    Figure Legend Snippet: Recognition of Cit-containing peptide epitope of fibrin β chain by antibodies in sera of RA patients and healthy blood donors a , b and by isolated B cells c . a Reactivities of RA ( n = 170) or healthy ( n = 138) serum samples with N-terminally bitotinylated β60-74Cit vs. β60-74Arg bound to neutravidin precoated plates. ELISA ratios were calculated (OD with β60-74Cit /OD with β60-74Arg). Data were analyzed with the Mann–Whitney test, and the median OD ratio of RA samples was 1.68, interquartile range 0.95–6.33, and the median OD ratio of healthy samples was 0.91, interquartile range 0.81–1.07 (*** p

    Techniques Used: Isolation, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    18) Product Images from "Binding of Recombinant Feline Immunodeficiency Virus Surface Glycoprotein to Feline Cells: Role of CXCR4, Cell-Surface Heparans, and an Unidentified Non-CXCR4 Receptor"

    Article Title: Binding of Recombinant Feline Immunodeficiency Virus Surface Glycoprotein to Feline Cells: Role of CXCR4, Cell-Surface Heparans, and an Unidentified Non-CXCR4 Receptor

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.10.4528-4539.2001

    gp95-Fc interacts with a 40-kDa protein species from the surface of feline primary T cells but not from Jurkat and 3201 cells. Cells were cell surface biotinylated and lysed, and precleared lysate supernatants were incubated with either Fc (lane 1), PPR gp95-Fc (PP; lane 2), or 34TF10 gp95-Fc (34; lane 3). Complexes were immunoprecipitated with protein A, resolved by SDS-PAGE, and immunoblotted with neutravidin. Bound neutravidin was revealed by an enhanced chemiluminescence procedure.
    Figure Legend Snippet: gp95-Fc interacts with a 40-kDa protein species from the surface of feline primary T cells but not from Jurkat and 3201 cells. Cells were cell surface biotinylated and lysed, and precleared lysate supernatants were incubated with either Fc (lane 1), PPR gp95-Fc (PP; lane 2), or 34TF10 gp95-Fc (34; lane 3). Complexes were immunoprecipitated with protein A, resolved by SDS-PAGE, and immunoblotted with neutravidin. Bound neutravidin was revealed by an enhanced chemiluminescence procedure.

    Techniques Used: Incubation, Immunoprecipitation, SDS Page

    19) Product Images from "Munc18 and Munc13 serve as a functional template to orchestrate neuronal SNARE complex assembly"

    Article Title: Munc18 and Munc13 serve as a functional template to orchestrate neuronal SNARE complex assembly

    Journal: Nature Communications

    doi: 10.1038/s41467-018-08028-6

    Syb2/MUN interaction promotes membrane association. a Illustration of the single-vesicle tethering assay. Glass surfaces were modified by PEG and biotin-PEG mixtures. DiD-labeled PM-vesicles containing 0.5% biotin-PE were first immobilized on a surface treated with neutravidin. After extensive washing, 1 μM Munc13-1 C 1 -C 2 B-MUN fragment (M13) and 40 μM (total lipids) Dil-labeled SV-vesicles were added and incubated for 30 min at 30 °C. Finally, another washing step was performed before imaging. b Representative channel image using Syb2 WT -bearing SV-vesicles and wild-type Munc13-1 C 1 -C 2 B-MUN fragment (M13). c Example channel image using Syb2 RKAA -bearing SV-vesicles and wild-type Munc13-1 C 1 -C 2 B-MUN fragment (M13). d Example channel image using Syb2 2WD -bearing SV-vesicles and wild-type Munc13-1 C 1 -C 2 B-MUN fragment (M13). e Example channel image using Syb2 4M -bearing SV-vesicles and wild-type Munc13-1 C 1 -C 2 B-MUN fragment (M13). f Example channel image using plain SV-vesicles and wild -type Munc13-1 C 1 -C 2 B-MUN fragment (M13). g Example channel image using Syb2 WT -bearing SV-vesicle and Munc13-1 C 1 -C 2 B-MUN fragment (M13) D1358K mutant. h Example channel image using Syb2 WT -bearing SV-vesicles only. i Quantification of the results in b – f . j Quantification of the results in b , f – h . Data are presented as the means ± SEM with dots showing individual single-vesicle counts from 10 randomly chosen frames ( n = 10). ** p
    Figure Legend Snippet: Syb2/MUN interaction promotes membrane association. a Illustration of the single-vesicle tethering assay. Glass surfaces were modified by PEG and biotin-PEG mixtures. DiD-labeled PM-vesicles containing 0.5% biotin-PE were first immobilized on a surface treated with neutravidin. After extensive washing, 1 μM Munc13-1 C 1 -C 2 B-MUN fragment (M13) and 40 μM (total lipids) Dil-labeled SV-vesicles were added and incubated for 30 min at 30 °C. Finally, another washing step was performed before imaging. b Representative channel image using Syb2 WT -bearing SV-vesicles and wild-type Munc13-1 C 1 -C 2 B-MUN fragment (M13). c Example channel image using Syb2 RKAA -bearing SV-vesicles and wild-type Munc13-1 C 1 -C 2 B-MUN fragment (M13). d Example channel image using Syb2 2WD -bearing SV-vesicles and wild-type Munc13-1 C 1 -C 2 B-MUN fragment (M13). e Example channel image using Syb2 4M -bearing SV-vesicles and wild-type Munc13-1 C 1 -C 2 B-MUN fragment (M13). f Example channel image using plain SV-vesicles and wild -type Munc13-1 C 1 -C 2 B-MUN fragment (M13). g Example channel image using Syb2 WT -bearing SV-vesicle and Munc13-1 C 1 -C 2 B-MUN fragment (M13) D1358K mutant. h Example channel image using Syb2 WT -bearing SV-vesicles only. i Quantification of the results in b – f . j Quantification of the results in b , f – h . Data are presented as the means ± SEM with dots showing individual single-vesicle counts from 10 randomly chosen frames ( n = 10). ** p

    Techniques Used: Modification, Labeling, Incubation, Imaging, Mutagenesis

    20) Product Images from "Action of the Chaperonin GroEL/ES on a Non-Native Substrate Observed with Single-Molecule FRET"

    Article Title: Action of the Chaperonin GroEL/ES on a Non-Native Substrate Observed with Single-Molecule FRET

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2010.06.050

    (Bottom) A specific binding test was performed using biotin- and Cy3-labeled GroEL (C473Bio-EL). In the presence of Neutravidin (left panel), Cy3-labeled C473Bio-EL could be
    Figure Legend Snippet: (Bottom) A specific binding test was performed using biotin- and Cy3-labeled GroEL (C473Bio-EL). In the presence of Neutravidin (left panel), Cy3-labeled C473Bio-EL could be

    Techniques Used: Binding Assay, Labeling

    21) Product Images from "Quantitative Determination of Size and Shape of Surface-Bound DNA Using an Acoustic Wave Sensor"

    Article Title: Quantitative Determination of Size and Shape of Surface-Bound DNA Using an Acoustic Wave Sensor

    Journal: Biophysical Journal

    doi: 10.1529/biophysj.107.119271

    Representation of the biosensor device surface/liquid interface: straight dsDNA molecules of various lengths are attached to the neutravidin-modified gold surface through a biotin linker incorporating an 11-carbon hinge. Three of the six different lengths used in this work are shown in the picture drawn using the GSVIEW software package (not drawn to scale). The white shade surrounding the DNA chains indicates the hydration layer of each molecule.
    Figure Legend Snippet: Representation of the biosensor device surface/liquid interface: straight dsDNA molecules of various lengths are attached to the neutravidin-modified gold surface through a biotin linker incorporating an 11-carbon hinge. Three of the six different lengths used in this work are shown in the picture drawn using the GSVIEW software package (not drawn to scale). The white shade surrounding the DNA chains indicates the hydration layer of each molecule.

    Techniques Used: Modification, Software

    Real-time binding curve of amplitude and phase change during the application of neutravidin (not shown) followed by ( a ) 198-bp dsDNA sample of 5 μ g/ml and ( b ) 250 μ g/ml of histone Hv1. Amplitude and phase are depicted with a dotted and solid line, respectively. Also shown is a schematic representation of the DNA configuration on the surface before and after the addition of the histone.
    Figure Legend Snippet: Real-time binding curve of amplitude and phase change during the application of neutravidin (not shown) followed by ( a ) 198-bp dsDNA sample of 5 μ g/ml and ( b ) 250 μ g/ml of histone Hv1. Amplitude and phase are depicted with a dotted and solid line, respectively. Also shown is a schematic representation of the DNA configuration on the surface before and after the addition of the histone.

    Techniques Used: Binding Assay

    Real-time binding curve of amplitude and phase change during the application of ( a ) neutravidin 100 μ g/ml, followed by 167-bp DNA samples of ( b ) 1.2, ( c ) 2.4, ( d ) 3.6, and ( e ) 4.8 μ g/ml. Buffer-washing steps following each deposition are not shown in the graph. Amplitude and phase are depicted with a dotted and solid line, respectively. ( Inset ) Acoustic ratio of amplitude change versus phase change (ΔA/ΔPh) measured for each DNA addition.
    Figure Legend Snippet: Real-time binding curve of amplitude and phase change during the application of ( a ) neutravidin 100 μ g/ml, followed by 167-bp DNA samples of ( b ) 1.2, ( c ) 2.4, ( d ) 3.6, and ( e ) 4.8 μ g/ml. Buffer-washing steps following each deposition are not shown in the graph. Amplitude and phase are depicted with a dotted and solid line, respectively. ( Inset ) Acoustic ratio of amplitude change versus phase change (ΔA/ΔPh) measured for each DNA addition.

    Techniques Used: Binding Assay

    22) Product Images from "Dynamics of human telomerase recruitment depend on template-telomere base pairing"

    Article Title: Dynamics of human telomerase recruitment depend on template-telomere base pairing

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E17-11-0637

    Imetelstat (GRN163L) is a competitive inhibitor of primer-substrate binding by telomerase. (A) Experimental design of single-molecule telomerase primer binding and activity assay. Halo-telomerase is modified with a biotin-HaloTag-ligand and immobilized on the coverslip surface using NeutrAvidin. Primer binding is visualized by telomerase-dependent recruitment of a fluorescent primer to the coverslip surface. The telomerase extension product is detected using a fluorescently labeled oligonucleotide anti-sense to the telomerase extension product. (B) Western blot and fluorescence imaging of Halo-telomerase modified with a fluorescent dye (JF646) or biotin, probed with an anti-TERT antibody or HRP-conjugated streptavidin. (C) Single-molecule TIRF imaging of primer molecules recruited to the coverslip surface by telomerase (top) and its colocalization with telomerase extension products after incubation with nucleotide substrate (bottom). (D) Single-molecule TIRF imaging of primer binding by telomerase in the presence of increasing concentrations of imetelstat. (E) Quantification of primer binding to telomerase as a function of imetelstat concentration ( n = 5 fields of view per concentration, data points plotted as mean ± SD, error on IC 50 reflects error in the corresponding fit of the data to a simple binding curve). (F) Direct telomerase assay at 150 mM KCl in the absence and presence of imetelstat (10 nM), or mismatched control oligonucleotide (MM Control, 10 nM), and increasing concentrations of primer substrate. LC1, LC2, and LC3, labeled DNA loading controls. (G) Quantification of telomerase activity as a function of primer concentration in absence and presence of imetelstat (10 nM) or mismatched control oligonucleotide (MM Control, 10 nM).
    Figure Legend Snippet: Imetelstat (GRN163L) is a competitive inhibitor of primer-substrate binding by telomerase. (A) Experimental design of single-molecule telomerase primer binding and activity assay. Halo-telomerase is modified with a biotin-HaloTag-ligand and immobilized on the coverslip surface using NeutrAvidin. Primer binding is visualized by telomerase-dependent recruitment of a fluorescent primer to the coverslip surface. The telomerase extension product is detected using a fluorescently labeled oligonucleotide anti-sense to the telomerase extension product. (B) Western blot and fluorescence imaging of Halo-telomerase modified with a fluorescent dye (JF646) or biotin, probed with an anti-TERT antibody or HRP-conjugated streptavidin. (C) Single-molecule TIRF imaging of primer molecules recruited to the coverslip surface by telomerase (top) and its colocalization with telomerase extension products after incubation with nucleotide substrate (bottom). (D) Single-molecule TIRF imaging of primer binding by telomerase in the presence of increasing concentrations of imetelstat. (E) Quantification of primer binding to telomerase as a function of imetelstat concentration ( n = 5 fields of view per concentration, data points plotted as mean ± SD, error on IC 50 reflects error in the corresponding fit of the data to a simple binding curve). (F) Direct telomerase assay at 150 mM KCl in the absence and presence of imetelstat (10 nM), or mismatched control oligonucleotide (MM Control, 10 nM), and increasing concentrations of primer substrate. LC1, LC2, and LC3, labeled DNA loading controls. (G) Quantification of telomerase activity as a function of primer concentration in absence and presence of imetelstat (10 nM) or mismatched control oligonucleotide (MM Control, 10 nM).

    Techniques Used: Binding Assay, Activity Assay, Modification, Labeling, Western Blot, Fluorescence, Imaging, Incubation, Concentration Assay, Telomerase Assay

    23) Product Images from "Sensing Reversible Protein–Ligand Interactions with Single-Walled Carbon Nanotube Field-Effect Transistors"

    Article Title: Sensing Reversible Protein–Ligand Interactions with Single-Walled Carbon Nanotube Field-Effect Transistors

    Journal: The Journal of Physical Chemistry. C, Nanomaterials and Interfaces

    doi: 10.1021/jp503670a

    Transistor characteristics of a biotinylated SWNT FET before (black) and after (red) NeutrAvidin adsorption, measured at pH 3.4 (a) and at pH 6.3 (b). (c) Transfer characteristics of an unfunctionalized device before (black) and after (red) NeutrAvidin adsorption measured at pH 6.5. (d) Normalized sensor response ( I 0 – I P )/ g m of pyrene-biotin-functionalized SWNT FET toward NeutrAvidin (blue) and streptavidin (gray) under different buffer concentrations as a function of buffer pH. The protein response was measured in 1.6–2 mM buffer (triangles) and 0.8–0.9 mM buffer (circles) concentrations. Error bars result from averaging responses of several devices. The solid lines represent sigmoidal Boltzmann fits.
    Figure Legend Snippet: Transistor characteristics of a biotinylated SWNT FET before (black) and after (red) NeutrAvidin adsorption, measured at pH 3.4 (a) and at pH 6.3 (b). (c) Transfer characteristics of an unfunctionalized device before (black) and after (red) NeutrAvidin adsorption measured at pH 6.5. (d) Normalized sensor response ( I 0 – I P )/ g m of pyrene-biotin-functionalized SWNT FET toward NeutrAvidin (blue) and streptavidin (gray) under different buffer concentrations as a function of buffer pH. The protein response was measured in 1.6–2 mM buffer (triangles) and 0.8–0.9 mM buffer (circles) concentrations. Error bars result from averaging responses of several devices. The solid lines represent sigmoidal Boltzmann fits.

    Techniques Used: Adsorption

    24) Product Images from "Enhanced Capture and Release of Circulating Tumor Cells Using Hollow Glass Microspheres with Nanostructured Surface"

    Article Title: Enhanced Capture and Release of Circulating Tumor Cells Using Hollow Glass Microspheres with Nanostructured Surface

    Journal: Nanoscale

    doi: 10.1039/c8nr04434a

    Fluorescence image and cross-sectional intensity profiles of LbL film on the outmost surface of NS HGMS before (a) and after (b) degradation by ALG lyase. Standard intensity was quantified using ImageJ and the fluorescence intensity of NS HGMS before degradation was set to 1.0. The biotin groups on NS HGMS surface were tagged with Neutravidin-Texas Red. SEM images of surface morphologies of NS HGMS before (c) and after (d) degradation by ALG lyase.
    Figure Legend Snippet: Fluorescence image and cross-sectional intensity profiles of LbL film on the outmost surface of NS HGMS before (a) and after (b) degradation by ALG lyase. Standard intensity was quantified using ImageJ and the fluorescence intensity of NS HGMS before degradation was set to 1.0. The biotin groups on NS HGMS surface were tagged with Neutravidin-Texas Red. SEM images of surface morphologies of NS HGMS before (c) and after (d) degradation by ALG lyase.

    Techniques Used: Fluorescence

    25) Product Images from "Retroviral intasomes search for a target DNA by 1D diffusion which rarely results in integration"

    Article Title: Retroviral intasomes search for a target DNA by 1D diffusion which rarely results in integration

    Journal: Nature Communications

    doi: 10.1038/ncomms11409

    PFV intasomes search a target DNA by 1D diffusion. ( a ) Experimental system for PFV intasome interaction with a target DNA using smTIRF imaging. The total internal reflection fluorescence (TIRF) field extends 200–250 nm into the flow cell (teal background). A target DNA is stretched by hydrodynamic flow and bound at both ends by the biotin–NeutrAvidin over the surface. The PFV intasomes were reconstituted with Cy3-labelled viral U5 cDNA. When the Cy3-labelled PFV intasome interacts with the target DNA inside the TIRF field, the fluorophore is excited allowing visual tracking of particles. ( b ) Representative trace of PFV intasome diffusion (yellow) on a target DNA (red) at a 300-ms frame rate. ( c ) Distribution of lifetimes (±s.e.) for wild type PFV intasomes at different NaCl salt concentrations ( Supplementary Fig. 6 ). ( d ) Box plots of the diffusion coefficients distribution for wild type PFV intasomes at different NaCl salt concentrations at a 100-ms frame rate ( Supplementary Fig. 6 ). Box plots show the mean (indentation), median (black line), upper and lower quartile (box ends), and outliers (whiskers).
    Figure Legend Snippet: PFV intasomes search a target DNA by 1D diffusion. ( a ) Experimental system for PFV intasome interaction with a target DNA using smTIRF imaging. The total internal reflection fluorescence (TIRF) field extends 200–250 nm into the flow cell (teal background). A target DNA is stretched by hydrodynamic flow and bound at both ends by the biotin–NeutrAvidin over the surface. The PFV intasomes were reconstituted with Cy3-labelled viral U5 cDNA. When the Cy3-labelled PFV intasome interacts with the target DNA inside the TIRF field, the fluorophore is excited allowing visual tracking of particles. ( b ) Representative trace of PFV intasome diffusion (yellow) on a target DNA (red) at a 300-ms frame rate. ( c ) Distribution of lifetimes (±s.e.) for wild type PFV intasomes at different NaCl salt concentrations ( Supplementary Fig. 6 ). ( d ) Box plots of the diffusion coefficients distribution for wild type PFV intasomes at different NaCl salt concentrations at a 100-ms frame rate ( Supplementary Fig. 6 ). Box plots show the mean (indentation), median (black line), upper and lower quartile (box ends), and outliers (whiskers).

    Techniques Used: Diffusion-based Assay, Imaging, Fluorescence, Flow Cytometry, Mass Spectrometry

    26) Product Images from "Retroviral intasomes search for a target DNA by 1D diffusion which rarely results in integration"

    Article Title: Retroviral intasomes search for a target DNA by 1D diffusion which rarely results in integration

    Journal: Nature Communications

    doi: 10.1038/ncomms11409

    PFV intasomes search a target DNA by 1D diffusion. ( a ) Experimental system for PFV intasome interaction with a target DNA using smTIRF imaging. The total internal reflection fluorescence (TIRF) field extends 200–250 nm into the flow cell (teal background). A target DNA is stretched by hydrodynamic flow and bound at both ends by the biotin–NeutrAvidin over the surface. The PFV intasomes were reconstituted with Cy3-labelled viral U5 cDNA. When the Cy3-labelled PFV intasome interacts with the target DNA inside the TIRF field, the fluorophore is excited allowing visual tracking of particles. ( b ) Representative trace of PFV intasome diffusion (yellow) on a target DNA (red) at a 300-ms frame rate. ( c ) Distribution of lifetimes (±s.e.) for wild type PFV intasomes at different NaCl salt concentrations ( Supplementary Fig. 6 ). ( d ) Box plots of the diffusion coefficients distribution for wild type PFV intasomes at different NaCl salt concentrations at a 100-ms frame rate ( Supplementary Fig. 6 ). Box plots show the mean (indentation), median (black line), upper and lower quartile (box ends), and outliers (whiskers).
    Figure Legend Snippet: PFV intasomes search a target DNA by 1D diffusion. ( a ) Experimental system for PFV intasome interaction with a target DNA using smTIRF imaging. The total internal reflection fluorescence (TIRF) field extends 200–250 nm into the flow cell (teal background). A target DNA is stretched by hydrodynamic flow and bound at both ends by the biotin–NeutrAvidin over the surface. The PFV intasomes were reconstituted with Cy3-labelled viral U5 cDNA. When the Cy3-labelled PFV intasome interacts with the target DNA inside the TIRF field, the fluorophore is excited allowing visual tracking of particles. ( b ) Representative trace of PFV intasome diffusion (yellow) on a target DNA (red) at a 300-ms frame rate. ( c ) Distribution of lifetimes (±s.e.) for wild type PFV intasomes at different NaCl salt concentrations ( Supplementary Fig. 6 ). ( d ) Box plots of the diffusion coefficients distribution for wild type PFV intasomes at different NaCl salt concentrations at a 100-ms frame rate ( Supplementary Fig. 6 ). Box plots show the mean (indentation), median (black line), upper and lower quartile (box ends), and outliers (whiskers).

    Techniques Used: Diffusion-based Assay, Imaging, Fluorescence, Flow Cytometry, Mass Spectrometry

    27) Product Images from "Fission yeast myosin Myo2 is down-regulated in actin affinity by light chain phosphorylation"

    Article Title: Fission yeast myosin Myo2 is down-regulated in actin affinity by light chain phosphorylation

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1703161114

    RLC phosphorylation reduces the number of Myo2 heads bound to actin filaments. Panels show maximum projections of representative fields (128 μm × 128 μm × 50 s) at different KCl concentrations ( A – C ). Phosphorylated Myo2 is shown on the Left (red) and unphosphorylated is shown on the Right ) are shown as averages of multiple fields (N, number of fields). Myo2 was attached to neutravidin-coated coverslips through the C-terminal biotin tag. Conditions were as follows: 30 °C, 25 mM imidazole, pH 7.4, KCl as indicated, 4 mM MgCl 2 , 1 mM EGTA, 1 mM ATP, and 10 mM DTT. No methylcellulose was used to allow the diffusion of actin away from the surface. When indicated, Tpm was added to a final concentration of 2 μM. Data are from two independent preparations of Myo2 (N-FLAG-C-Biotin). Error, ± SD.
    Figure Legend Snippet: RLC phosphorylation reduces the number of Myo2 heads bound to actin filaments. Panels show maximum projections of representative fields (128 μm × 128 μm × 50 s) at different KCl concentrations ( A – C ). Phosphorylated Myo2 is shown on the Left (red) and unphosphorylated is shown on the Right ) are shown as averages of multiple fields (N, number of fields). Myo2 was attached to neutravidin-coated coverslips through the C-terminal biotin tag. Conditions were as follows: 30 °C, 25 mM imidazole, pH 7.4, KCl as indicated, 4 mM MgCl 2 , 1 mM EGTA, 1 mM ATP, and 10 mM DTT. No methylcellulose was used to allow the diffusion of actin away from the surface. When indicated, Tpm was added to a final concentration of 2 μM. Data are from two independent preparations of Myo2 (N-FLAG-C-Biotin). Error, ± SD.

    Techniques Used: Diffusion-based Assay, Concentration Assay

    Effects of RLC phosphorylation on ATPase activity and motility. ( A ) Steady-state ATPase of unphosphorylated (blue) or phosphorylated (red) Myo2 in the absence or presence of tropomyosin (Tpm). Circles and squares represent independent preparations of N-FLAG-Myo2-C-Biotin. Diamonds represent N-FLAG-Myo2. Conditions were as follows: 30 °C, 10 mM imidazole, pH 7.0, 50 mM NaCl, 1 mM MgCl 2 , 1 mM ATP, and 2 mM DTT. Tpm was added at a 2:1 Actin-Tpm molar ratio. ( B ) In vitro motility speeds of unphosphorylated or phosphorylated Myo2, in the absence or presence of Tpm. Speeds represent motility data from two experimental repeats conducted in parallel with two independent preparations of N-FLAG-Myo2-C-Biotin (n, number of moving filaments). To ensure that all heads were available for interaction with actin, Myo2 was attached to neutravidin-coated coverslips by a biotin tag at its C terminus. Conditions were as follows: 30 °C, 0.5% methylcellulose, 25 mM imidazole, pH 7.4, 50 mM KCl, 4 mM MgCl 2 , 1 mM EGTA, 1 mM ATP, and 10 mM DTT. When indicated, Tpm was added to a final concentration of 2 μM.
    Figure Legend Snippet: Effects of RLC phosphorylation on ATPase activity and motility. ( A ) Steady-state ATPase of unphosphorylated (blue) or phosphorylated (red) Myo2 in the absence or presence of tropomyosin (Tpm). Circles and squares represent independent preparations of N-FLAG-Myo2-C-Biotin. Diamonds represent N-FLAG-Myo2. Conditions were as follows: 30 °C, 10 mM imidazole, pH 7.0, 50 mM NaCl, 1 mM MgCl 2 , 1 mM ATP, and 2 mM DTT. Tpm was added at a 2:1 Actin-Tpm molar ratio. ( B ) In vitro motility speeds of unphosphorylated or phosphorylated Myo2, in the absence or presence of Tpm. Speeds represent motility data from two experimental repeats conducted in parallel with two independent preparations of N-FLAG-Myo2-C-Biotin (n, number of moving filaments). To ensure that all heads were available for interaction with actin, Myo2 was attached to neutravidin-coated coverslips by a biotin tag at its C terminus. Conditions were as follows: 30 °C, 0.5% methylcellulose, 25 mM imidazole, pH 7.4, 50 mM KCl, 4 mM MgCl 2 , 1 mM EGTA, 1 mM ATP, and 10 mM DTT. When indicated, Tpm was added to a final concentration of 2 μM.

    Techniques Used: Activity Assay, In Vitro, Concentration Assay

    28) Product Images from "Constructing 3D microtubule networks using holographic optical trapping"

    Article Title: Constructing 3D microtubule networks using holographic optical trapping

    Journal: Scientific Reports

    doi: 10.1038/srep18085

    Basic BH-MT tethering strategy and preparation of assay constituents. ( a ) Motorized cargos are functionalized with WT hKIF5A while bead handles are functionalized with non-motile mutant hKIF5A (E237A). Construct sequences for kinesin-1 are otherwise identical, consisting of full length KHC and a HIS6 tag at the C-terminus. ( b ) BHs are specifically bound to E237A hKIF5A via a biotin-neutravidin-biotin sandwich (B-N-B in the figure) functionalized with a Nickel-activated tris-NTA molecule that binds with high affinity to the HIS6 tag on the E237A hKIF5A construct. Protein structure shows kinesin-1 motor domain. E237A mutation is highlighted in red and is in close proximity with a nucleotide (cyan). ( c , d ) BHs and MCs are prepared separately (see Methods), then combined together with MTs in assay buffer and finally are introduced into a flow chamber to allow filament network assembly.
    Figure Legend Snippet: Basic BH-MT tethering strategy and preparation of assay constituents. ( a ) Motorized cargos are functionalized with WT hKIF5A while bead handles are functionalized with non-motile mutant hKIF5A (E237A). Construct sequences for kinesin-1 are otherwise identical, consisting of full length KHC and a HIS6 tag at the C-terminus. ( b ) BHs are specifically bound to E237A hKIF5A via a biotin-neutravidin-biotin sandwich (B-N-B in the figure) functionalized with a Nickel-activated tris-NTA molecule that binds with high affinity to the HIS6 tag on the E237A hKIF5A construct. Protein structure shows kinesin-1 motor domain. E237A mutation is highlighted in red and is in close proximity with a nucleotide (cyan). ( c , d ) BHs and MCs are prepared separately (see Methods), then combined together with MTs in assay buffer and finally are introduced into a flow chamber to allow filament network assembly.

    Techniques Used: Mutagenesis, Construct, Flow Cytometry

    29) Product Images from "Aptamer-phage reporters for ultrasensitive lateral flow assays"

    Article Title: Aptamer-phage reporters for ultrasensitive lateral flow assays

    Journal: Analytical chemistry

    doi: 10.1021/acs.analchem.5b00702

    Functionalization of M13 phage with IgE aptamers and Horseradish peroxidase (HRP). Phage displaying AviTag peptides are biotinylated using Biotin ligase. Biotinylated phage are covalently modified with HRP on the major coat protein pVIII before neutravidin
    Figure Legend Snippet: Functionalization of M13 phage with IgE aptamers and Horseradish peroxidase (HRP). Phage displaying AviTag peptides are biotinylated using Biotin ligase. Biotinylated phage are covalently modified with HRP on the major coat protein pVIII before neutravidin

    Techniques Used: Modification

    30) Product Images from "MUC1 Aptamer Targeted SERS Nanoprobes"

    Article Title: MUC1 Aptamer Targeted SERS Nanoprobes

    Journal: Advanced functional materials

    doi: 10.1002/adfm.201606632

    (a) Synthesis and DNA functionalization of SERS NPs. First, 70 nm spherical AuNP cores were silica coated in the presence of IR dye (IR 780 perchlorate or IR 792 perchlorate) to produce SERS NPs. The SERS NPs were functionalized with DNA molecules by sequential modification of the silica shell surface. To verify the presence of the DNA sequence, a 10 nm AuNP functionalized with the complementary DNA strand was hybridized to produce SERS NP core - 10 nm AuNP satellite hybrid nanostructures. (b) Illustration of DNA functionalization: first, the surface hydroxyl groups were converted to sulfhydryl groups using (3-mercaptopropyl)trimethoxysilane (MPTMS) in a water-ethanol mixture. Next, sulfhydryl groups were reacted with a maleimide-PEG 2 -biotin linker to attach biotin molecules to the surface. Subsequently, biotin labeled DNA molecules were attached to the biotinylated surface of the SERS NPs using neutravidin, a biotin-binding protein as a crosslinker. (c) Representative TEM images of the DNA functionalized SERS NPs. (d) The DLS hydrodynamic diameter measurements show an increase in size from ~80 nm to ~137 nm after silica coating and ~170 nm after DNA functionalization. Error bars represent standard deviations of three measurements. (e) Representative TEM images of the SERS NP core – 10nm AuNP satellite hybrid nanostructures. The inset shows 10 nm AuNP satellites being attached to the SERS NP surface. All the scale bars are 100 nm.
    Figure Legend Snippet: (a) Synthesis and DNA functionalization of SERS NPs. First, 70 nm spherical AuNP cores were silica coated in the presence of IR dye (IR 780 perchlorate or IR 792 perchlorate) to produce SERS NPs. The SERS NPs were functionalized with DNA molecules by sequential modification of the silica shell surface. To verify the presence of the DNA sequence, a 10 nm AuNP functionalized with the complementary DNA strand was hybridized to produce SERS NP core - 10 nm AuNP satellite hybrid nanostructures. (b) Illustration of DNA functionalization: first, the surface hydroxyl groups were converted to sulfhydryl groups using (3-mercaptopropyl)trimethoxysilane (MPTMS) in a water-ethanol mixture. Next, sulfhydryl groups were reacted with a maleimide-PEG 2 -biotin linker to attach biotin molecules to the surface. Subsequently, biotin labeled DNA molecules were attached to the biotinylated surface of the SERS NPs using neutravidin, a biotin-binding protein as a crosslinker. (c) Representative TEM images of the DNA functionalized SERS NPs. (d) The DLS hydrodynamic diameter measurements show an increase in size from ~80 nm to ~137 nm after silica coating and ~170 nm after DNA functionalization. Error bars represent standard deviations of three measurements. (e) Representative TEM images of the SERS NP core – 10nm AuNP satellite hybrid nanostructures. The inset shows 10 nm AuNP satellites being attached to the SERS NP surface. All the scale bars are 100 nm.

    Techniques Used: Modification, Sequencing, Labeling, Binding Assay, Transmission Electron Microscopy

    31) Product Images from "Nanoparticle Enhancement Cascade for Sensitive Multiplex Measurements of Biomarkers in Complex Fluids with Surface Plasmon Resonance Imaging"

    Article Title: Nanoparticle Enhancement Cascade for Sensitive Multiplex Measurements of Biomarkers in Complex Fluids with Surface Plasmon Resonance Imaging

    Journal: Analytical Chemistry

    doi: 10.1021/acs.analchem.8b00260

    Lower limit of detection is plotted against the affinity of the capture antibody for the four cytokines measured. The figure shows good correlation between the capture antibody affinity and the LLOD in a power law function ( R 2 = 0.98). This indicates that the capture antibody affinity is the main predictor for the sensitivity of the assay. Variations in detection antibody affinity and biotin availability for neutravidin potentially play a less pronounced role.
    Figure Legend Snippet: Lower limit of detection is plotted against the affinity of the capture antibody for the four cytokines measured. The figure shows good correlation between the capture antibody affinity and the LLOD in a power law function ( R 2 = 0.98). This indicates that the capture antibody affinity is the main predictor for the sensitivity of the assay. Variations in detection antibody affinity and biotin availability for neutravidin potentially play a less pronounced role.

    Techniques Used:

    Cytokines were measured specifically in multiplex. (A) The RU signal during gold nanoparticle association after interaction with a mixture of cytokines in the lower region of the dynamic detection range, followed by the detection antibody and neutravidin cascade. The graph shows the baseline, followed by 15 min of association and 10 min of dissociation. The signals shown are an average of 10 spots. (B–E) Similar measurements as in (A) were performed; however, the concentration of a single cytokine is increased to the higher region in their dynamic detection range, without changing other concentrations in the mixture. In this way, the specificity and cross-reactivity of the cytokine detection was assessed.
    Figure Legend Snippet: Cytokines were measured specifically in multiplex. (A) The RU signal during gold nanoparticle association after interaction with a mixture of cytokines in the lower region of the dynamic detection range, followed by the detection antibody and neutravidin cascade. The graph shows the baseline, followed by 15 min of association and 10 min of dissociation. The signals shown are an average of 10 spots. (B–E) Similar measurements as in (A) were performed; however, the concentration of a single cytokine is increased to the higher region in their dynamic detection range, without changing other concentrations in the mixture. In this way, the specificity and cross-reactivity of the cytokine detection was assessed.

    Techniques Used: Multiplex Assay, Concentration Assay

    SPRi enhancement cascade response over time. The interaction with IL-6 is shown, followed by specific detection antibody, neutravidin, and gold nanoparticle. The graph (A) shows the average signal, in refractive index units (RU), combined from eight unique antibody spots with a uniform spotting density, corrected as described to R max = 100 RU. The subplots (B–E) show the individual autoscaled graphs for each step in the signal enhancement cascade.
    Figure Legend Snippet: SPRi enhancement cascade response over time. The interaction with IL-6 is shown, followed by specific detection antibody, neutravidin, and gold nanoparticle. The graph (A) shows the average signal, in refractive index units (RU), combined from eight unique antibody spots with a uniform spotting density, corrected as described to R max = 100 RU. The subplots (B–E) show the individual autoscaled graphs for each step in the signal enhancement cascade.

    Techniques Used:

    Enhancement cascade leads to sequential improvement in sensitivity and dynamic detection range. (A) The signal increase (dRU) after association with analyte, detection antibody (Det. Ab), neutravidin (NeuAv), and gold nanoparticle (GNP) is shown for IL-6 over a concentration range from 100 fg/mL to 100 ng/mL (1 μg/mL is omitted due to clipping of too high signal). The inset shows the linear signal increase between 0.1 and 100 pg/mL ( R 2 . The signals represent the average of eight spots, corrected to R max = 100 RU. Error bars depict standard deviations. Four-parameter logistic regression was used to fit the data points. (B–D) The signal after gold nanoparticle enhancement for IL-1β, IFN-γ, and TNF-α over a concentration range from 100 fg/mL to 1 μg/mL.
    Figure Legend Snippet: Enhancement cascade leads to sequential improvement in sensitivity and dynamic detection range. (A) The signal increase (dRU) after association with analyte, detection antibody (Det. Ab), neutravidin (NeuAv), and gold nanoparticle (GNP) is shown for IL-6 over a concentration range from 100 fg/mL to 100 ng/mL (1 μg/mL is omitted due to clipping of too high signal). The inset shows the linear signal increase between 0.1 and 100 pg/mL ( R 2 . The signals represent the average of eight spots, corrected to R max = 100 RU. Error bars depict standard deviations. Four-parameter logistic regression was used to fit the data points. (B–D) The signal after gold nanoparticle enhancement for IL-1β, IFN-γ, and TNF-α over a concentration range from 100 fg/mL to 1 μg/mL.

    Techniques Used: Concentration Assay

    SPRi signal enhancement cascade using biotinylated gold nanoparticles. In this cascade, there is a sequential buildup of the complex and thus in SPRi signal. Initially, a specific capture antibody (1) interacts with the antigen (2); this is followed by a specific detection antibody toward that antigen in a sandwich format (3). Subsequently, neutravidin binds to the complex (4) followed by a commercially available biotinylated gold nanoparticle for large signal improvement (5).
    Figure Legend Snippet: SPRi signal enhancement cascade using biotinylated gold nanoparticles. In this cascade, there is a sequential buildup of the complex and thus in SPRi signal. Initially, a specific capture antibody (1) interacts with the antigen (2); this is followed by a specific detection antibody toward that antigen in a sandwich format (3). Subsequently, neutravidin binds to the complex (4) followed by a commercially available biotinylated gold nanoparticle for large signal improvement (5).

    Techniques Used:

    Spikes in synovial fluid can be measured with only minor variation compared to buffer controls. (A) The graph shows RU signal after interaction with a mixture of cytokines in the higher region of the dynamic detection range, followed by the detection antibody, neutravidin, and gold nanoparticle cascade. The cytokines were spiked in synovial fluid, in half synovial fluid and half buffer, or in pure buffer. The signals shown represent the average of 10 measurement spots. (B–E) Subplots are shown for each step in the enhancement cascade. The graphs show the baseline, followed by association and dissociation.
    Figure Legend Snippet: Spikes in synovial fluid can be measured with only minor variation compared to buffer controls. (A) The graph shows RU signal after interaction with a mixture of cytokines in the higher region of the dynamic detection range, followed by the detection antibody, neutravidin, and gold nanoparticle cascade. The cytokines were spiked in synovial fluid, in half synovial fluid and half buffer, or in pure buffer. The signals shown represent the average of 10 measurement spots. (B–E) Subplots are shown for each step in the enhancement cascade. The graphs show the baseline, followed by association and dissociation.

    Techniques Used:

    32) Product Images from "Lateral Membrane Diffusion Modulated by a Minimal Actin Cortex"

    Article Title: Lateral Membrane Diffusion Modulated by a Minimal Actin Cortex

    Journal: Biophysical Journal

    doi: 10.1016/j.bpj.2013.02.042

    Scheme of the MAC. The filamentous biotinylated ( blue ) actin is coupled via neutravidin to the free-standing membrane (Egg PC) containing biotinylated lipids (DSPE-PEG(2000)-Biotin). For a more compact display the binding of actin is shown only to the
    Figure Legend Snippet: Scheme of the MAC. The filamentous biotinylated ( blue ) actin is coupled via neutravidin to the free-standing membrane (Egg PC) containing biotinylated lipids (DSPE-PEG(2000)-Biotin). For a more compact display the binding of actin is shown only to the

    Techniques Used: Binding Assay

    33) Product Images from "Proteins modified by the lipid peroxidation aldehyde DODE in MCF7 breast cancer cells"

    Article Title: Proteins modified by the lipid peroxidation aldehyde DODE in MCF7 breast cancer cells

    Journal: Chemical research in toxicology

    doi: 10.1021/tx9002808

    2D-SDS-PAGE MCF7 proteins treated with ARP and affinity purified by neutravidin. A: Coomassie-blue stained gel. B: Anti-biotin Western blot.
    Figure Legend Snippet: 2D-SDS-PAGE MCF7 proteins treated with ARP and affinity purified by neutravidin. A: Coomassie-blue stained gel. B: Anti-biotin Western blot.

    Techniques Used: SDS Page, Affinity Purification, Staining, Western Blot

    34) Product Images from "Origin of nanomechanical cantilever motion generated from biomolecular interactions"

    Article Title: Origin of nanomechanical cantilever motion generated from biomolecular interactions

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Change in cantilever deflection on exposure to neutravidin. In one case, the silicon nitride surface of the cantilever was coated with biotin and polyethylene glycol, whereas in the other case, the same surface cantilever was coated with a silane layer. Specific binding of neutravidin to biotin generated a negative deflection, indicating generation of compressive stress on the top silicon nitride surface, whereas exposure to silane produced no appreciable deflection signal.
    Figure Legend Snippet: Change in cantilever deflection on exposure to neutravidin. In one case, the silicon nitride surface of the cantilever was coated with biotin and polyethylene glycol, whereas in the other case, the same surface cantilever was coated with a silane layer. Specific binding of neutravidin to biotin generated a negative deflection, indicating generation of compressive stress on the top silicon nitride surface, whereas exposure to silane produced no appreciable deflection signal.

    Techniques Used: Binding Assay, Generated, Produced

    35) Product Images from "Self-repair promotes microtubule rescue"

    Article Title: Self-repair promotes microtubule rescue

    Journal: Nature cell biology

    doi: 10.1038/ncb3406

    Microtubule self-repair induces rescue events in vitro. (a) Rescue at crossing microtubules. Time-lapse sequence of 3 microtubules crossing each other. The kymograph highlights the crossing sites (yellow arrow-head pointing at the bright white vertical lines) and the occurrence of multiple rescue events at this site (red arrow-heads). (b) The graph shows the frequency of rescue events for crossing microtubules as a function of distance from the crossing site. Data represent mean +/- s.d from n=8 independent experiments. (c) Repair at crossing microtubules. Observation of the incorporation of green tubulin dimers along red microtubules. White arrow-heads point at crossing sites where accumulation of green tubulin was detected. Image is representative of 3 independent experiments. Scale bar 5 µm. (d) Illustration of the microfluidic device. Short biotinylated microtubule seeds were fixed on neutravidin coated micropatterns and elongated using red or green free tubulin. To exchange or remove the solution of free tubulin, a flow was induced parallel to the microtubules. (e) Photo damage sites can induce rescue. The image sequences and kymographs show microtubule dynamics with (right) and without (left) laser-induced damage. The green arrows indicate the seed. Red arrow-heads indicate rescue events. (f) The graph shows the frequency of rescue events for photo damaged microtubules as a function of distance from the center of the damage (green bars) and for microtubules without damage as a distance from the center of the observed microtubule (magenta bars). Data represent mean values +/- s.d from n= 4 independent experiments. (g) Tubulin incorporation at photodamaged sites is associated with rescue. Green microtubule seeds were elongated with red free tubulin (step I). A GMPCPP cap was grown at the microtubule tip to avoid spontaneous depolymerization (step II). Photo damage was induced in the presence of green tubulin (step III). Depolymerization was initiated by removing the GMPCPP cap with a laser pulse at high intensity (step IV). The kymograph shows rescue (red arrow-head) at the damaged site where green tubulin was incorporated. Image is representative of 4 independent experiments.
    Figure Legend Snippet: Microtubule self-repair induces rescue events in vitro. (a) Rescue at crossing microtubules. Time-lapse sequence of 3 microtubules crossing each other. The kymograph highlights the crossing sites (yellow arrow-head pointing at the bright white vertical lines) and the occurrence of multiple rescue events at this site (red arrow-heads). (b) The graph shows the frequency of rescue events for crossing microtubules as a function of distance from the crossing site. Data represent mean +/- s.d from n=8 independent experiments. (c) Repair at crossing microtubules. Observation of the incorporation of green tubulin dimers along red microtubules. White arrow-heads point at crossing sites where accumulation of green tubulin was detected. Image is representative of 3 independent experiments. Scale bar 5 µm. (d) Illustration of the microfluidic device. Short biotinylated microtubule seeds were fixed on neutravidin coated micropatterns and elongated using red or green free tubulin. To exchange or remove the solution of free tubulin, a flow was induced parallel to the microtubules. (e) Photo damage sites can induce rescue. The image sequences and kymographs show microtubule dynamics with (right) and without (left) laser-induced damage. The green arrows indicate the seed. Red arrow-heads indicate rescue events. (f) The graph shows the frequency of rescue events for photo damaged microtubules as a function of distance from the center of the damage (green bars) and for microtubules without damage as a distance from the center of the observed microtubule (magenta bars). Data represent mean values +/- s.d from n= 4 independent experiments. (g) Tubulin incorporation at photodamaged sites is associated with rescue. Green microtubule seeds were elongated with red free tubulin (step I). A GMPCPP cap was grown at the microtubule tip to avoid spontaneous depolymerization (step II). Photo damage was induced in the presence of green tubulin (step III). Depolymerization was initiated by removing the GMPCPP cap with a laser pulse at high intensity (step IV). The kymograph shows rescue (red arrow-head) at the damaged site where green tubulin was incorporated. Image is representative of 4 independent experiments.

    Techniques Used: In Vitro, Sequencing, Flow Cytometry

    36) Product Images from "Fast sorting of CD4+ T cells from whole blood using glass microbubbles"

    Article Title: Fast sorting of CD4+ T cells from whole blood using glass microbubbles

    Journal: Technology

    doi: 10.1142/S2339547815500016

    Images of glass microbubbles labeled with PE-conjugated biotin. Unmodified glass microbubbles (as shown in the phase-contrast image a ) are not labeled with PE-conjugated biotin as there is no fluorescence shown in the fluorescence image b , where as the surface-modified glass microbubbles (as shown in the phase-contrast image c ) have bright ring-shaped fluorescence shown in image d , indicating that the PE-conjugated biotin molecules are immobilized on the surface of glass microbubbles by their NeutrAvidin coating (scale bar = 50 μm).
    Figure Legend Snippet: Images of glass microbubbles labeled with PE-conjugated biotin. Unmodified glass microbubbles (as shown in the phase-contrast image a ) are not labeled with PE-conjugated biotin as there is no fluorescence shown in the fluorescence image b , where as the surface-modified glass microbubbles (as shown in the phase-contrast image c ) have bright ring-shaped fluorescence shown in image d , indicating that the PE-conjugated biotin molecules are immobilized on the surface of glass microbubbles by their NeutrAvidin coating (scale bar = 50 μm).

    Techniques Used: Labeling, Fluorescence, Modification

    37) Product Images from "Single-Molecule Fluorescence Reveals the Unwinding Stepping Mechanism of Replicative Helicase"

    Article Title: Single-Molecule Fluorescence Reveals the Unwinding Stepping Mechanism of Replicative Helicase

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2014.02.022

    The Unwinding Rate of T7 Helicase Depends on Base Pair Stability (A) T7 gp4 was loaded on a 40 bp DNA with (dT)n tails containing donor (Cy3) and acceptor (Cy5) dyes, and bound to a PEG-coated surface via biotin-neutravidin interaction. (B) Cy3 and Cy5 intensity traces during unwinding for one molecule on mixed, 100% AT, and 80% GC sequences (top panel); calculated FRET efficiency versus time for the fluorescence intensity traces (bottom panel). (C) Dwell-time histograms during unwinding. The arrows on the FRET traces indicate the intervals at which the dwell times were measured; 50 molecules were used to build the histograms. The data are representative of multiple experiments. (D) Unwinding time (red circles) and rate (blue triangles) versus base pair stability. Five sequences were used to plot the graph ( Table S1 ). See also Figure S1 .
    Figure Legend Snippet: The Unwinding Rate of T7 Helicase Depends on Base Pair Stability (A) T7 gp4 was loaded on a 40 bp DNA with (dT)n tails containing donor (Cy3) and acceptor (Cy5) dyes, and bound to a PEG-coated surface via biotin-neutravidin interaction. (B) Cy3 and Cy5 intensity traces during unwinding for one molecule on mixed, 100% AT, and 80% GC sequences (top panel); calculated FRET efficiency versus time for the fluorescence intensity traces (bottom panel). (C) Dwell-time histograms during unwinding. The arrows on the FRET traces indicate the intervals at which the dwell times were measured; 50 molecules were used to build the histograms. The data are representative of multiple experiments. (D) Unwinding time (red circles) and rate (blue triangles) versus base pair stability. Five sequences were used to plot the graph ( Table S1 ). See also Figure S1 .

    Techniques Used: Fluorescence

    38) Product Images from "Peptide mimotopes of malondialdehyde epitopes for clinical applications in cardiovascular disease [S]"

    Article Title: Peptide mimotopes of malondialdehyde epitopes for clinical applications in cardiovascular disease [S]

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M025445

    Linear P1 and cyclic P2 peptides mimic a specific MDA epitope. (A, B) Schematic representation of synthesized peptides carrying the consensus sequence from phages identified with the Ph.D.-12 and Ph.D.-C7C libraries, respectively. P1(A) is a linear dodecameric peptide ( HSWTNSWMATFL ), and P2 (B) is a cyclic heptameric peptide (C NNSNMPL C). The peptides’ C terminus containing a GGGC-spacer was amidated. (C, D) ELISA for the binding of LRO4 to P1 and P2. (C) Binding of LRO4 to P1 and P2. Peptides P1, P2, and an irrelevant control peptide were coated at indicated concentrations. Binding of LRO4 (5 μg/ml) was determined by chemiluminescent ELISA. (D) Indicated concentrations of biotinylated peptides were captured on wells precoated with 10 μg/ml of neutravidin, and binding of LRO4 (5 μg/ml) was determined as described in Materials and Methods. Values are given as relative light units (RLU) per 100 ms and represent the mean ± SD of triplicate determinations. (E–G) Immunocompetition assays for the antigenic properties of P1 and P2. Binding of 0.5 μg/ml LRO4 to 100 ng/ml captured biotinylated peptides P1 (E) or P2 (F) or to 0.5 μg/ml coated MAA-BSA (G) was measured by ELISA in the presence of increasing concentrations of indicated competitors. Data represent the mean ± SD of triplicate determinations and are representative of three independent experiments.
    Figure Legend Snippet: Linear P1 and cyclic P2 peptides mimic a specific MDA epitope. (A, B) Schematic representation of synthesized peptides carrying the consensus sequence from phages identified with the Ph.D.-12 and Ph.D.-C7C libraries, respectively. P1(A) is a linear dodecameric peptide ( HSWTNSWMATFL ), and P2 (B) is a cyclic heptameric peptide (C NNSNMPL C). The peptides’ C terminus containing a GGGC-spacer was amidated. (C, D) ELISA for the binding of LRO4 to P1 and P2. (C) Binding of LRO4 to P1 and P2. Peptides P1, P2, and an irrelevant control peptide were coated at indicated concentrations. Binding of LRO4 (5 μg/ml) was determined by chemiluminescent ELISA. (D) Indicated concentrations of biotinylated peptides were captured on wells precoated with 10 μg/ml of neutravidin, and binding of LRO4 (5 μg/ml) was determined as described in Materials and Methods. Values are given as relative light units (RLU) per 100 ms and represent the mean ± SD of triplicate determinations. (E–G) Immunocompetition assays for the antigenic properties of P1 and P2. Binding of 0.5 μg/ml LRO4 to 100 ng/ml captured biotinylated peptides P1 (E) or P2 (F) or to 0.5 μg/ml coated MAA-BSA (G) was measured by ELISA in the presence of increasing concentrations of indicated competitors. Data represent the mean ± SD of triplicate determinations and are representative of three independent experiments.

    Techniques Used: Multiple Displacement Amplification, Synthesized, Sequencing, Enzyme-linked Immunosorbent Assay, Binding Assay, Chemiluminescent ELISA, Mass Spectrometry

    39) Product Images from "Development of a Peptide-Modified siRNA Nanocomplex for Hepatic Stellate Cells"

    Article Title: Development of a Peptide-Modified siRNA Nanocomplex for Hepatic Stellate Cells

    Journal: Nanomedicine : nanotechnology, biology, and medicine

    doi: 10.1016/j.nano.2017.08.017

    Characterization and silencing activity of the nanocomplex (A) Gel retardation assay of the SNCP, SNVP and SNPP nanocomplexes at different N/P ratios. (B) Zeta potential of the nanocomplexes with different N/P ratios. (C) Particle size of the nanocomplexes. (SNC: siRNA-neutravidin-cholesterol complex; SNV: siRNA-neutravidin- vitamin A complex; SNP: siRNA-neutravidin-peptide-431 complex). (D) Serum Stability of the nanocomplex in 50% rat serum for 0, 6, 12, and 24h. (E) Cytotoxicity study of the nanocomplexes.
    Figure Legend Snippet: Characterization and silencing activity of the nanocomplex (A) Gel retardation assay of the SNCP, SNVP and SNPP nanocomplexes at different N/P ratios. (B) Zeta potential of the nanocomplexes with different N/P ratios. (C) Particle size of the nanocomplexes. (SNC: siRNA-neutravidin-cholesterol complex; SNV: siRNA-neutravidin- vitamin A complex; SNP: siRNA-neutravidin-peptide-431 complex). (D) Serum Stability of the nanocomplex in 50% rat serum for 0, 6, 12, and 24h. (E) Cytotoxicity study of the nanocomplexes.

    Techniques Used: Activity Assay, Electrophoretic Mobility Shift Assay

    The synthesis and fabrication schemes of (A) biotin-conjugated cholesterol, (B) biotin-conjugated vitamin A, (C) biotin-conjugated IGF2R-speific peptide, and (D) the neutravidin-based siRNA nanocomplex Biotin-conjugated PCBP2 siRNA, neutravidin and biotin-conjugated ligands were mixed in a 2:1:2 ratio at room temperature to form the siRNA-neutravidin-ligand complex, followed by condensation with protamine to form the final nanocomplex
    Figure Legend Snippet: The synthesis and fabrication schemes of (A) biotin-conjugated cholesterol, (B) biotin-conjugated vitamin A, (C) biotin-conjugated IGF2R-speific peptide, and (D) the neutravidin-based siRNA nanocomplex Biotin-conjugated PCBP2 siRNA, neutravidin and biotin-conjugated ligands were mixed in a 2:1:2 ratio at room temperature to form the siRNA-neutravidin-ligand complex, followed by condensation with protamine to form the final nanocomplex

    Techniques Used:

    40) Product Images from "Endosomal Dynamics of Met Determine Signaling Output"

    Article Title: Endosomal Dynamics of Met Determine Signaling Output

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E02-09-0578

    Inhibition of the proteasome has no effect on ligand-induced Met internalization but instead promotes recycling to the plasma membrane. (A) Cell-surface receptors were biotinylated on ice with a disulfide-cleavable biotin as described in MATERIALS AND METHODS. Cells were then rapidly rewarmed to 37°C and cultured in medium with or without HGF/SF and/or proteasome inhibitor for the indicated times to allow internalization of surface Met, after which they were rapidly cooled on ice, and remaining cell-surface biotin stripped with MESNA. After lysis, internalized biotinylated molecules were recovered with NeutrAvidin-conjugated beads, resolved by SDS-PAGE, and examined by Western analysis under reducing conditions using anti-Met Hu intracellular-domain antibody. Similar results were obtained in three experiments. (B) To investigate recycling of Met after a 10-min HGF/SF “pulse,” cells were rewarmed after MESNA stripping, but in the absence of ligand, for 15 or 30 min at 37°C then subjected to a second round of MESNA stripping before lysis and analysis as described in A. Molecular mass markers are shown on the left.
    Figure Legend Snippet: Inhibition of the proteasome has no effect on ligand-induced Met internalization but instead promotes recycling to the plasma membrane. (A) Cell-surface receptors were biotinylated on ice with a disulfide-cleavable biotin as described in MATERIALS AND METHODS. Cells were then rapidly rewarmed to 37°C and cultured in medium with or without HGF/SF and/or proteasome inhibitor for the indicated times to allow internalization of surface Met, after which they were rapidly cooled on ice, and remaining cell-surface biotin stripped with MESNA. After lysis, internalized biotinylated molecules were recovered with NeutrAvidin-conjugated beads, resolved by SDS-PAGE, and examined by Western analysis under reducing conditions using anti-Met Hu intracellular-domain antibody. Similar results were obtained in three experiments. (B) To investigate recycling of Met after a 10-min HGF/SF “pulse,” cells were rewarmed after MESNA stripping, but in the absence of ligand, for 15 or 30 min at 37°C then subjected to a second round of MESNA stripping before lysis and analysis as described in A. Molecular mass markers are shown on the left.

    Techniques Used: Inhibition, Cell Culture, Lysis, SDS Page, Western Blot, Stripping Membranes

    Isolation of biotinylated Met: efficiency of MESNA reduction and optimization of cell-surface Met biotinylation. (A) HeLa cells were incubated on ice for 15 min ± 500 μg/ml disulfide-cleavable biotin, as described in MATERIALS AND METHODS. The cells were then washed of excess biotin by three quenching rinses and either lysed immediately (lane 2), or after three incubations in a solution containing the reducing agent MESNA (lane 3), plus additional washes. Surface biotinylated molecules were recovered with NeutrAvidin-agarose beads, resolved by 8% SDS-PAGE, and examined by Western blot analysis under reducing conditions using anti-Met Hu intracellular-domain antibody. MESNA efficiently cleaves all biotin-Met moieties. (B) Biotinylations were carried out with varying concentrations of biotin. Excess biotin was removed by three quenching rinses and the cells were then lysed. i) Surface biotinylated molecules were recovered with NeutrAvidin-agarose beads, resolved by SDS-PAGE, and examined by Western blot analysis under reducing conditions using anti-Met Hu intracellular-domain antibody. ii) The unbound fraction from each precipitation reaction was also probed with the same anti-Met Hu antibody. Molecular mass markers (in kilodaltons) are shown on the left. Using 250 μg/ml or more of biotin (lanes 5 and 6) results in only a very small (
    Figure Legend Snippet: Isolation of biotinylated Met: efficiency of MESNA reduction and optimization of cell-surface Met biotinylation. (A) HeLa cells were incubated on ice for 15 min ± 500 μg/ml disulfide-cleavable biotin, as described in MATERIALS AND METHODS. The cells were then washed of excess biotin by three quenching rinses and either lysed immediately (lane 2), or after three incubations in a solution containing the reducing agent MESNA (lane 3), plus additional washes. Surface biotinylated molecules were recovered with NeutrAvidin-agarose beads, resolved by 8% SDS-PAGE, and examined by Western blot analysis under reducing conditions using anti-Met Hu intracellular-domain antibody. MESNA efficiently cleaves all biotin-Met moieties. (B) Biotinylations were carried out with varying concentrations of biotin. Excess biotin was removed by three quenching rinses and the cells were then lysed. i) Surface biotinylated molecules were recovered with NeutrAvidin-agarose beads, resolved by SDS-PAGE, and examined by Western blot analysis under reducing conditions using anti-Met Hu intracellular-domain antibody. ii) The unbound fraction from each precipitation reaction was also probed with the same anti-Met Hu antibody. Molecular mass markers (in kilodaltons) are shown on the left. Using 250 μg/ml or more of biotin (lanes 5 and 6) results in only a very small (

    Techniques Used: Isolation, Incubation, SDS Page, Western Blot

    41) Product Images from "Characterizing exogenous mRNA delivery, trafficking, cytoplasmic release and RNA–protein correlations at the level of single cells"

    Article Title: Characterizing exogenous mRNA delivery, trafficking, cytoplasmic release and RNA–protein correlations at the level of single cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx290

    mRNA labeling, validation, and transfection into cells using cationic lipids or electroporation. ( A ) Illustrative diagram of mRNA labeling and delivery. MTRIPS are composed of four biotinylated and fluorescently labeled oligonucleotides assembled on a Neutravidin core. They bind to IVT mRNA in the 3΄ UTR region. Fluorescently labeled mRNA is electroporated or lipofected into cells. Electroporated mRNA is immediately available in the cytosol, while lipofected mRNA must first travel through endosomes until released. Degradation and sequestration of mRNA reduces final expression of the target protein. Highlighted mechanisms of importance include (i) mRNA entry pathway, (ii) cytoplasmic release, (iii) translational efficiency (RNA/protein correlation) and (iv) innate immune (PKR) activation. ( B ) Labeled mRNA (green) delivered via lipofection colocalized with Stellaris FISH probe signal (red). Line profiles are indicated by white lines. No FISH signal was visible in cells transfected with an mRNA containing a different coding region. (scale bar = 10 μm). ( C ) Cationic lipid transfection and electroporation with labeled mRNA encoding EGFP resulted in similar levels of protein production (green) but distinct subcellular distributions of mRNA (red) at 2 h post-transfection. Upon electroporation, small mRNA granules were observed throughout the cell cytoplasm, while cationic lipid transfection resulted in few large mRNA granules (scale bar = 20 μm). ( D ) Flow cytometry of EGFP expression using labeled versus unlabeled EGFP mRNA showed only a slight reduction in protein expression.
    Figure Legend Snippet: mRNA labeling, validation, and transfection into cells using cationic lipids or electroporation. ( A ) Illustrative diagram of mRNA labeling and delivery. MTRIPS are composed of four biotinylated and fluorescently labeled oligonucleotides assembled on a Neutravidin core. They bind to IVT mRNA in the 3΄ UTR region. Fluorescently labeled mRNA is electroporated or lipofected into cells. Electroporated mRNA is immediately available in the cytosol, while lipofected mRNA must first travel through endosomes until released. Degradation and sequestration of mRNA reduces final expression of the target protein. Highlighted mechanisms of importance include (i) mRNA entry pathway, (ii) cytoplasmic release, (iii) translational efficiency (RNA/protein correlation) and (iv) innate immune (PKR) activation. ( B ) Labeled mRNA (green) delivered via lipofection colocalized with Stellaris FISH probe signal (red). Line profiles are indicated by white lines. No FISH signal was visible in cells transfected with an mRNA containing a different coding region. (scale bar = 10 μm). ( C ) Cationic lipid transfection and electroporation with labeled mRNA encoding EGFP resulted in similar levels of protein production (green) but distinct subcellular distributions of mRNA (red) at 2 h post-transfection. Upon electroporation, small mRNA granules were observed throughout the cell cytoplasm, while cationic lipid transfection resulted in few large mRNA granules (scale bar = 20 μm). ( D ) Flow cytometry of EGFP expression using labeled versus unlabeled EGFP mRNA showed only a slight reduction in protein expression.

    Techniques Used: Labeling, Transfection, Electroporation, Expressing, Activation Assay, Fluorescence In Situ Hybridization, Flow Cytometry, Cytometry

    42) Product Images from "Computing in mammalian cells with nucleic acid strand exchange"

    Article Title: Computing in mammalian cells with nucleic acid strand exchange

    Journal: Nature nanotechnology

    doi: 10.1038/nnano.2015.278

    Endogenous mRNA and multiply-labeled, tetravalent imaging probes (mMTRIPS) can serve as scaffolds for strand exchange reactions a. i. An mRNA targeting oligo is covalently linked to NeutrAvidin (Sigma) via an aromatic hydrazine and aldehyde linkage (Hynic-4FB, Solulink), with neutravidin naturally forming tetramers. ii. One oligo making up the 2′OMe RNA reporter has an additional poly-T linker and biotin moiety at the 5′ end. Note that to increase brightness and photostability for the microscopy measurements, Cy3b/BHQ2 fluorophore/quencher pair was utilized instead of the TYE665/IAB. iii. The biotin-labeled gate associates with the NeutrAvidin tetramer to form a multiply-labeled, tetravalent imaging probe (mMTRIP). iv-v. mMTRIPS targeting five separate sites on the β-actin mRNA (ACTB) were introduced into A549 cells using SLO delivery (15 nM each). Following an hour-long recovery period, the indicated input complex was introduced at a 1x or 3x molar excess (75 or 225 nM, respectively) using SLO delivery. 4-way strand exchange reactions were visualized using mMTRIPs scaffolded on endogenous ACTB mRNAs ( b ) and un-targeted mMTRIPs ( c ). i. Details of the reactants. ii. Representative epifluorescence microscopy images. Images were deconvolved and analyzed to quantify the fluorescence signal of individual puncta and the mean fluorescence intensity was determined. iii. Mean fluorescence intensity, normalized to the quenched reporter. * indicates statistically different data according to Kruskal-Wallis One Way Analysis of Variance on Ranks. Scale bars are 10 μm. Error bars indicate one standard deviation.
    Figure Legend Snippet: Endogenous mRNA and multiply-labeled, tetravalent imaging probes (mMTRIPS) can serve as scaffolds for strand exchange reactions a. i. An mRNA targeting oligo is covalently linked to NeutrAvidin (Sigma) via an aromatic hydrazine and aldehyde linkage (Hynic-4FB, Solulink), with neutravidin naturally forming tetramers. ii. One oligo making up the 2′OMe RNA reporter has an additional poly-T linker and biotin moiety at the 5′ end. Note that to increase brightness and photostability for the microscopy measurements, Cy3b/BHQ2 fluorophore/quencher pair was utilized instead of the TYE665/IAB. iii. The biotin-labeled gate associates with the NeutrAvidin tetramer to form a multiply-labeled, tetravalent imaging probe (mMTRIP). iv-v. mMTRIPS targeting five separate sites on the β-actin mRNA (ACTB) were introduced into A549 cells using SLO delivery (15 nM each). Following an hour-long recovery period, the indicated input complex was introduced at a 1x or 3x molar excess (75 or 225 nM, respectively) using SLO delivery. 4-way strand exchange reactions were visualized using mMTRIPs scaffolded on endogenous ACTB mRNAs ( b ) and un-targeted mMTRIPs ( c ). i. Details of the reactants. ii. Representative epifluorescence microscopy images. Images were deconvolved and analyzed to quantify the fluorescence signal of individual puncta and the mean fluorescence intensity was determined. iii. Mean fluorescence intensity, normalized to the quenched reporter. * indicates statistically different data according to Kruskal-Wallis One Way Analysis of Variance on Ranks. Scale bars are 10 μm. Error bars indicate one standard deviation.

    Techniques Used: Labeling, Imaging, Microscopy, Epifluorescence Microscopy, Fluorescence, Standard Deviation

    43) Product Images from "Diffusive tail anchorage determines velocity and force produced by kinesin-14 between crosslinked microtubules"

    Article Title: Diffusive tail anchorage determines velocity and force produced by kinesin-14 between crosslinked microtubules

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04656-0

    Diffusively anchored kinesin-14 motors exert sub-pN forces. a Sparsely cy5-labeled, digoxigeninated microtubules were surface immobilized using anti-digoxigenin antibodies. The surface was passivated using the polymer pluronic F127. Full-length Ncd (flNcd) motors crosslinked surface-immobilized microtubules and transported microtubules, which were densely cy5-labeled and biotinylated (sliding assay geometry). A NeutrAvidin-coated microsphere bound to the transported microtubule was manipulated using optical tweezers. b Velocity distribution of microtubules transported by flNcd ( N = 265). c Velocities of transported microtubules without external load were constant over a wide range of microtubule lengths. The number of microtubules per depicted bin is shown in Supplementary Figure 2 . For the boxplot elements description see Methods. d Characteristic force trace for a microsphere attached to a microtubule that is transported by flNcd. Note that once the microsphere attaches to the microtubule, sliding motion stops immediately and the microsphere is not transported out of the trap center. Direction of microtubule sliding is parallel to the x- axis. The binding event is marked with a red arrow. The last movement of the microscope stage prior to binding is marked with a vertical red line. e Typical force-velocity measurement used to quantify the stopping force of flNcd in microtubule overlaps. Below, a schematic zoom-in is shown. f Stopping force distribution of 33 independent transported microtubules (for method of force quantification see Fig. 2e). g Force distribution of 49 independent microtubule pairs estimated from the microtubule overlap length and the number of Ase1 molecules within that overlap. Inset: Depiction of the assay used to determine this force distribution. The non-enzymatic, diffusive crosslinker Ase1, when confined in a microtubule overlap, exerts an entropic force resisting its compaction in the shortening microtubule overlap. Ncd-driven microtubule sliding stops when force balance is reached 19 . h Schematic depiction of the microtubule buckling assay. Two sets of microtubules were surface immobilized using anti-digoxigenin antibodies: dimly rhodamine-labeled digoxigeninated and cy5-labeled, biotinylated, digoxigeninated microtubules. NeutrAvidin was used to coat the biotinylated microtubules. flNcd was added and biotinylated, brightly rhodamine-labeled transport microtubules were allowed to bind. Upon encountering NeutrAvidin the transported microtubule stopped and, if Ncd exerted sufficient force, buckled. i Micrographs showing the buckling event with the highest force, which was calculated to be 1.1 pN. Several tens of seconds of thermal fluctuation preceded the buckling; scale bar 5 µm
    Figure Legend Snippet: Diffusively anchored kinesin-14 motors exert sub-pN forces. a Sparsely cy5-labeled, digoxigeninated microtubules were surface immobilized using anti-digoxigenin antibodies. The surface was passivated using the polymer pluronic F127. Full-length Ncd (flNcd) motors crosslinked surface-immobilized microtubules and transported microtubules, which were densely cy5-labeled and biotinylated (sliding assay geometry). A NeutrAvidin-coated microsphere bound to the transported microtubule was manipulated using optical tweezers. b Velocity distribution of microtubules transported by flNcd ( N = 265). c Velocities of transported microtubules without external load were constant over a wide range of microtubule lengths. The number of microtubules per depicted bin is shown in Supplementary Figure 2 . For the boxplot elements description see Methods. d Characteristic force trace for a microsphere attached to a microtubule that is transported by flNcd. Note that once the microsphere attaches to the microtubule, sliding motion stops immediately and the microsphere is not transported out of the trap center. Direction of microtubule sliding is parallel to the x- axis. The binding event is marked with a red arrow. The last movement of the microscope stage prior to binding is marked with a vertical red line. e Typical force-velocity measurement used to quantify the stopping force of flNcd in microtubule overlaps. Below, a schematic zoom-in is shown. f Stopping force distribution of 33 independent transported microtubules (for method of force quantification see Fig. 2e). g Force distribution of 49 independent microtubule pairs estimated from the microtubule overlap length and the number of Ase1 molecules within that overlap. Inset: Depiction of the assay used to determine this force distribution. The non-enzymatic, diffusive crosslinker Ase1, when confined in a microtubule overlap, exerts an entropic force resisting its compaction in the shortening microtubule overlap. Ncd-driven microtubule sliding stops when force balance is reached 19 . h Schematic depiction of the microtubule buckling assay. Two sets of microtubules were surface immobilized using anti-digoxigenin antibodies: dimly rhodamine-labeled digoxigeninated and cy5-labeled, biotinylated, digoxigeninated microtubules. NeutrAvidin was used to coat the biotinylated microtubules. flNcd was added and biotinylated, brightly rhodamine-labeled transport microtubules were allowed to bind. Upon encountering NeutrAvidin the transported microtubule stopped and, if Ncd exerted sufficient force, buckled. i Micrographs showing the buckling event with the highest force, which was calculated to be 1.1 pN. Several tens of seconds of thermal fluctuation preceded the buckling; scale bar 5 µm

    Techniques Used: Labeling, Binding Assay, Microscopy

    44) Product Images from "Sensitive Detection of Norovirus Using Phage Nanoparticle Reporters in Lateral-Flow Assay"

    Article Title: Sensitive Detection of Norovirus Using Phage Nanoparticle Reporters in Lateral-Flow Assay

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0126571

    Detection of Norwalk VLPs using a sandwich ELISA. A) Antibody pair screening for the detection of Norwalk VLPs; values correspond to the absorbance for a sample for 10 9 VLPs offered; background absorbance for no VLP sample was subtracted (typical value ~0.1). Red color denotes maximum ΔOD450 observed in the ELISA, yellow lowest, and a smooth color gradient in between. Black box denotes the sandwich pair that was used in LFA. B) Sandwich ELISA detecting Norwalk VLPs where F2 was used as the capturing antibody. For the detection biotinylated F1 and streptavidin HRP (antibody sandwich, closed symbols), or the phage construct (Antibody-NeutrAvidin-AviTag phage) and anti-M13/ HRP conjugate (phage sandwich; open symbols) were used.
    Figure Legend Snippet: Detection of Norwalk VLPs using a sandwich ELISA. A) Antibody pair screening for the detection of Norwalk VLPs; values correspond to the absorbance for a sample for 10 9 VLPs offered; background absorbance for no VLP sample was subtracted (typical value ~0.1). Red color denotes maximum ΔOD450 observed in the ELISA, yellow lowest, and a smooth color gradient in between. Black box denotes the sandwich pair that was used in LFA. B) Sandwich ELISA detecting Norwalk VLPs where F2 was used as the capturing antibody. For the detection biotinylated F1 and streptavidin HRP (antibody sandwich, closed symbols), or the phage construct (Antibody-NeutrAvidin-AviTag phage) and anti-M13/ HRP conjugate (phage sandwich; open symbols) were used.

    Techniques Used: Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Construct

    45) Product Images from "Generation of Recombinant Antibodies to Rat GABAA Receptor Subunits by Affinity Selection on Synthetic Peptides"

    Article Title: Generation of Recombinant Antibodies to Rat GABAA Receptor Subunits by Affinity Selection on Synthetic Peptides

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0087964

    Binding of phage-expressed scFvs affinity selected with GABA subunit peptides. Equal amounts of biotinylated target peptides or non-target peptide (negative control) were captured on NeutrAvidin™ (NA) coated microtiter plate wells, and after washing the binding of equivalent amounts of phage particles, displaying different scFvs, was monitored by ELISA. A biotinylated anti-Flag antibody was used to normalize the amounts of scFv-displaying phage particles added to each well. Error bars correspond to the standard deviation of triplicate measurements of the optical density of the wells at 405 nm wavelength. A7, G8 and G11 are anti-β2 binders while A10 is the anti-α1 binder.
    Figure Legend Snippet: Binding of phage-expressed scFvs affinity selected with GABA subunit peptides. Equal amounts of biotinylated target peptides or non-target peptide (negative control) were captured on NeutrAvidin™ (NA) coated microtiter plate wells, and after washing the binding of equivalent amounts of phage particles, displaying different scFvs, was monitored by ELISA. A biotinylated anti-Flag antibody was used to normalize the amounts of scFv-displaying phage particles added to each well. Error bars correspond to the standard deviation of triplicate measurements of the optical density of the wells at 405 nm wavelength. A7, G8 and G11 are anti-β2 binders while A10 is the anti-α1 binder.

    Techniques Used: Binding Assay, Negative Control, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Comparison of detection limits of scFv and scFv-Fc. A. Binding specificity of equimolar concentrations of scFv-Fc and scFv A10, to different biotinylated peptides was detected in an ELISA using anti-Flag antibody conjugated to HRP. Signal intensities were measured at 405 nm and error bars indicate standard deviation of duplicate trials. NA, NeutrAvidin™; negative control pep, retinal protein Pep 1 peptide; beta 2, GABA A receptor subunit β2 peptide; rho 1, GABA c receptor subunit ρ pep tide and alpha 1, GABA A receptor subunit α1 peptide. B. Dot blots of varying concentrations of MBP-α1 proteins treated with 10 nM A10 scFv-Fc (top row) and scFv (bottom row). Circles around signal spots indicate pencil markings of protein addition sites. C. Signal intensities normalized to highest signal obtained for each trail are plotted against log of concentration for scFv-Fc and scFv. Error bars depict standard deviations. n = 3.
    Figure Legend Snippet: Comparison of detection limits of scFv and scFv-Fc. A. Binding specificity of equimolar concentrations of scFv-Fc and scFv A10, to different biotinylated peptides was detected in an ELISA using anti-Flag antibody conjugated to HRP. Signal intensities were measured at 405 nm and error bars indicate standard deviation of duplicate trials. NA, NeutrAvidin™; negative control pep, retinal protein Pep 1 peptide; beta 2, GABA A receptor subunit β2 peptide; rho 1, GABA c receptor subunit ρ pep tide and alpha 1, GABA A receptor subunit α1 peptide. B. Dot blots of varying concentrations of MBP-α1 proteins treated with 10 nM A10 scFv-Fc (top row) and scFv (bottom row). Circles around signal spots indicate pencil markings of protein addition sites. C. Signal intensities normalized to highest signal obtained for each trail are plotted against log of concentration for scFv-Fc and scFv. Error bars depict standard deviations. n = 3.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Negative Control, Concentration Assay

    46) Product Images from "TRBP ensures efficient Dicer processing of precursor microRNA in RNA-crowded environments"

    Article Title: TRBP ensures efficient Dicer processing of precursor microRNA in RNA-crowded environments

    Journal: Nature Communications

    doi: 10.1038/ncomms13694

    TRBP's dsRBDs mediate the recruitment of pre-miRNA in an RNA-crowded environment. ( a ) Schematic of sample preparation. Dicer was constructed with Flag, TEV and AP tags, which were used for immunoprecipitation, elution and biotinylation, respectively. Dicer proteins were biotinylated in HEK 293 cells. The proteins were immunoprecipitated using Flag-antibody beads. The proteins were eluted out of the beads via TEV cleavage. ( b ) Western blotting of Dicer IPs. Dicer proteins were expressed without and with Myc-TRBP and were pulled down using Flag beads (left). In vitro cleavage of pre-let-7a-1 3′2nt (right). Mock is a negative control with Flag-mCherry IPs. ( c ) Schematic of single-molecule immobilization. Dicer IPs were conjugated to a polymer-coated surface via NeutrAvidin–biotin interaction. Contaminant proteins were washed away before 200 pM Cy5-labelled pre-let-7a-1 3′2nt was introduced. Interactions between the surface-immobilized Dicer complexes with Cy5-labelled pre-miRNA were visualized through total internal reflection fluorescence (TIRF) microscopy. Dots in the CCD (charge-coupled device) image reflect docking of pre-let-7a-1 3′2nt to individual Dicer complexes. The CCD image illustrated the binding events over 25 × 25 μm 2 field of view. Scale bar, 5 μm. ( d ) The CCD images in the left illustrate the stable docking of Cy-5-labelled pre-let-7a-1 3′2nt to surface-immobilized Dicer complexes in absence (upper panels) and presence of 1 μM competitor tRNA (bottom panels). The histogram in the right quantifies the inhibition of the pre-miRNA-binding activity due to the presence of 1 μM competitor tRNA. Error is the s.d. obtained from 10 different fields of view in three independent experiments. Scale bar, 5 μm.
    Figure Legend Snippet: TRBP's dsRBDs mediate the recruitment of pre-miRNA in an RNA-crowded environment. ( a ) Schematic of sample preparation. Dicer was constructed with Flag, TEV and AP tags, which were used for immunoprecipitation, elution and biotinylation, respectively. Dicer proteins were biotinylated in HEK 293 cells. The proteins were immunoprecipitated using Flag-antibody beads. The proteins were eluted out of the beads via TEV cleavage. ( b ) Western blotting of Dicer IPs. Dicer proteins were expressed without and with Myc-TRBP and were pulled down using Flag beads (left). In vitro cleavage of pre-let-7a-1 3′2nt (right). Mock is a negative control with Flag-mCherry IPs. ( c ) Schematic of single-molecule immobilization. Dicer IPs were conjugated to a polymer-coated surface via NeutrAvidin–biotin interaction. Contaminant proteins were washed away before 200 pM Cy5-labelled pre-let-7a-1 3′2nt was introduced. Interactions between the surface-immobilized Dicer complexes with Cy5-labelled pre-miRNA were visualized through total internal reflection fluorescence (TIRF) microscopy. Dots in the CCD (charge-coupled device) image reflect docking of pre-let-7a-1 3′2nt to individual Dicer complexes. The CCD image illustrated the binding events over 25 × 25 μm 2 field of view. Scale bar, 5 μm. ( d ) The CCD images in the left illustrate the stable docking of Cy-5-labelled pre-let-7a-1 3′2nt to surface-immobilized Dicer complexes in absence (upper panels) and presence of 1 μM competitor tRNA (bottom panels). The histogram in the right quantifies the inhibition of the pre-miRNA-binding activity due to the presence of 1 μM competitor tRNA. Error is the s.d. obtained from 10 different fields of view in three independent experiments. Scale bar, 5 μm.

    Techniques Used: Sample Prep, Construct, Immunoprecipitation, Western Blot, In Vitro, Negative Control, Fluorescence, Microscopy, Binding Assay, Inhibition, Activity Assay

    TRBP binds dsRNA without selectivity. ( a ) Schematic representation of a single-molecule assay to capture RNA recognition by TRBP in real time. TRBP was immobilized on a surface via a biotinylated anti-glutathione- S -transferase (GST) antibody. ( b – d ) Representative time traces (a time resolution 500 ms) that exhibit binding of Cy5-labelled RNA to a single TRBP protein. The histograms (right) indicate the distribution of the dwell time. The dwell time is the time between docking and dissociation. ( e ) A representative time trace from Cy5-labelled pre-let-7a-1 3′2nt-biotin , which is immobilized on the surface via biotin–NeutrAvidin conjugation. The dwell time reflects the timescale of photobleaching. ( f ) Average dwell times from b to e . Error is the s.d. of four independent experiments.
    Figure Legend Snippet: TRBP binds dsRNA without selectivity. ( a ) Schematic representation of a single-molecule assay to capture RNA recognition by TRBP in real time. TRBP was immobilized on a surface via a biotinylated anti-glutathione- S -transferase (GST) antibody. ( b – d ) Representative time traces (a time resolution 500 ms) that exhibit binding of Cy5-labelled RNA to a single TRBP protein. The histograms (right) indicate the distribution of the dwell time. The dwell time is the time between docking and dissociation. ( e ) A representative time trace from Cy5-labelled pre-let-7a-1 3′2nt-biotin , which is immobilized on the surface via biotin–NeutrAvidin conjugation. The dwell time reflects the timescale of photobleaching. ( f ) Average dwell times from b to e . Error is the s.d. of four independent experiments.

    Techniques Used: Mass Spectrometry, Binding Assay, Conjugation Assay

    47) Product Images from "Time-resolved microscopy for imaging lanthanide luminescence in living cells"

    Article Title: Time-resolved microscopy for imaging lanthanide luminescence in living cells

    Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

    doi: 10.1002/cyto.a.20964

    Spectra, luminescent lifetime, and photobleaching kinetics of europium microspheres used in this study. (a) Normalized excitation (dotted) and emission spectra (solid) of 40 nm, NeutrAvidin ® -labeled europium microspheres (Fluospheres ™
    Figure Legend Snippet: Spectra, luminescent lifetime, and photobleaching kinetics of europium microspheres used in this study. (a) Normalized excitation (dotted) and emission spectra (solid) of 40 nm, NeutrAvidin ® -labeled europium microspheres (Fluospheres ™

    Techniques Used: Labeling

    48) Product Images from "Leuko-polymersomes"

    Article Title: Leuko-polymersomes

    Journal: Faraday discussions

    doi:

    Left: chemical scheme for biotinylatyion of 4F3 NB functionalized polymer. Upper right: giant biotinylated vesicles with NeutrAvidin, primary (biotinylated) antibody, and secondary antibody associated imaged using HDIC microscopy. Lower right: NeutrAvidin-coated
    Figure Legend Snippet: Left: chemical scheme for biotinylatyion of 4F3 NB functionalized polymer. Upper right: giant biotinylated vesicles with NeutrAvidin, primary (biotinylated) antibody, and secondary antibody associated imaged using HDIC microscopy. Lower right: NeutrAvidin-coated

    Techniques Used: Microscopy

    Flow cytometry of vesicles bearing anti-ICAM-1. Negative controls were vesicles coated with NeutrAvidin, and then labeled with biotinylated goat anti-mouse IgG antibody (left column). Positive controls employed a biotinylated murine anti-human ICAM-1
    Figure Legend Snippet: Flow cytometry of vesicles bearing anti-ICAM-1. Negative controls were vesicles coated with NeutrAvidin, and then labeled with biotinylated goat anti-mouse IgG antibody (left column). Positive controls employed a biotinylated murine anti-human ICAM-1

    Techniques Used: Flow Cytometry, Cytometry, Labeling

    49) Product Images from "Proteomic and bioinformatic pipeline to screen the ligands of S. pneumoniae interacting with human brain microvascular endothelial cells"

    Article Title: Proteomic and bioinformatic pipeline to screen the ligands of S. pneumoniae interacting with human brain microvascular endothelial cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-23485-1

    Overview of experimental and bioinformatic pipeline proposed in the study. Proteome of pneumococci was biotinylated and incubated with human brain microvascular cells (BMEC). Multiple interactions among various labeled proteins and surface of BMEC are represented as [1, 2, 3… n]. Potentially interacting ligands recovered with NeutrAvidin capture beads were identified by SWATH-MS. A systematic bioinformatic workflow was established for the proper selection of candidates for recombinant production and further validation of the proposed scheme. First, proteins were categorized based on the subcellular location, which followed by application of featured/based algorithms to retrieve detailed information like presence of transmembrane domains, prediction of signal peptide etc. Third, selection of proteins with high probability to bind host cells based on surface exposure. Fourth, characterization of candidates based on the ontology analysis, data search in protein repositories and literature. As a last bioinformatics analysis step, prediction of interactive proteins was performed to assess role of proteins in the process of pathogenesis. Finally, ligand candidates were selected for recombinant production and the proposed workflow was validated with ELISA. Selected ligands were evaluated in silico for antigenicity and immunogenicity. The proposed workflow was validated with ELISA and immunocytochemistry using recombinant form of ligands.
    Figure Legend Snippet: Overview of experimental and bioinformatic pipeline proposed in the study. Proteome of pneumococci was biotinylated and incubated with human brain microvascular cells (BMEC). Multiple interactions among various labeled proteins and surface of BMEC are represented as [1, 2, 3… n]. Potentially interacting ligands recovered with NeutrAvidin capture beads were identified by SWATH-MS. A systematic bioinformatic workflow was established for the proper selection of candidates for recombinant production and further validation of the proposed scheme. First, proteins were categorized based on the subcellular location, which followed by application of featured/based algorithms to retrieve detailed information like presence of transmembrane domains, prediction of signal peptide etc. Third, selection of proteins with high probability to bind host cells based on surface exposure. Fourth, characterization of candidates based on the ontology analysis, data search in protein repositories and literature. As a last bioinformatics analysis step, prediction of interactive proteins was performed to assess role of proteins in the process of pathogenesis. Finally, ligand candidates were selected for recombinant production and the proposed workflow was validated with ELISA. Selected ligands were evaluated in silico for antigenicity and immunogenicity. The proposed workflow was validated with ELISA and immunocytochemistry using recombinant form of ligands.

    Techniques Used: Incubation, Labeling, Mass Spectrometry, Selection, Recombinant, Enzyme-linked Immunosorbent Assay, In Silico, Immunocytochemistry

    Biotin labeling of proteome of pneumococci and confirmation of the presence of biotinylated proteins bound to human BMEC. Panel A shows protein labeling. Lane 1, protein extract of pneumococci prior to biotinylation separated on SDS-PAGE. Lane 2, biotinylated proteins were incubated on NeutrAvidin capture beads, eluted with 50 mM DTT and separated on SDS-PAGE. Lane 1 and 2 were cropped from original image obtained after PAGE (see Supplementary Figure 5 ). Note that the gel depicted here is representative of four replicates. Panel B shows the presence of biotinylated proteins in S2.Protein extract of BMEC obtained after incubation of biotinylated proteins of pneumococci with BMEC was spotted on the membrane and detected with IRdye®800 Streptavidin (S2) in dot blot. Total protein extract of human BMECs was spotted on membrane and incubated with IRdye®800 Streptavidin (negative control); Biotinylated proteins of pneumococci were spotted on membrane and detected with IRdye®800 Streptavidin (input control). ( B ) was created by combining cropped fragments from two membranes (see Supplementary Figure 6 ).
    Figure Legend Snippet: Biotin labeling of proteome of pneumococci and confirmation of the presence of biotinylated proteins bound to human BMEC. Panel A shows protein labeling. Lane 1, protein extract of pneumococci prior to biotinylation separated on SDS-PAGE. Lane 2, biotinylated proteins were incubated on NeutrAvidin capture beads, eluted with 50 mM DTT and separated on SDS-PAGE. Lane 1 and 2 were cropped from original image obtained after PAGE (see Supplementary Figure 5 ). Note that the gel depicted here is representative of four replicates. Panel B shows the presence of biotinylated proteins in S2.Protein extract of BMEC obtained after incubation of biotinylated proteins of pneumococci with BMEC was spotted on the membrane and detected with IRdye®800 Streptavidin (S2) in dot blot. Total protein extract of human BMECs was spotted on membrane and incubated with IRdye®800 Streptavidin (negative control); Biotinylated proteins of pneumococci were spotted on membrane and detected with IRdye®800 Streptavidin (input control). ( B ) was created by combining cropped fragments from two membranes (see Supplementary Figure 6 ).

    Techniques Used: Labeling, SDS Page, Incubation, Polyacrylamide Gel Electrophoresis, Dot Blot, Negative Control

    50) Product Images from "Strong Clonal Relatedness between Serum and Gut IgA despite Different Plasma Cell Origins"

    Article Title: Strong Clonal Relatedness between Serum and Gut IgA despite Different Plasma Cell Origins

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2017.08.036

    Detection of Antigen-Specific Serum Antibodies and Gut PCs (A) Serum antibody reactivity against TG2 or DGP in patients with untreated celiac disease (UCD, n = 12) or control subjects (n = 12) as determined by ELISA. Sera were diluted 1:200 and added to biotinylated TG2 or biotinylated DGP (biotin-GSGSGS-PLQPEQPFP, harboring an immunodominant gliadin epitope) ( Schwertz et al., 2004 , Steinsbø et al., 2014 ) immobilized on streptavidin followed by detection of bound IgA or IgG using isotype-specific goat anti-human antibodies. The dynamic range of the assay was comparable for detection of the different antibodies. Open symbols indicate subjects from whom serum antibodies were purified for further characterization. The same symbols are used throughout this study. (B) Correlation between the levels of serum IgA and IgG against each of the two antigens in celiac disease patients (n = 20). Reactivity was measured by ELISA, and the signals for each antigen are given relative to the IgA signal obtained with a reference serum sample (UCD1283). (C) ELISPOT detection of total or antigen-specific antibody-secreting cells (ASCs) producing IgA or IgG in gut biopsy specimens obtained from celiac disease patients (n = 8). Cell suspensions were added to coated NeutrAvidin alone or NeutrAvidin associated with biotinylated TG2 or DGP as indicated. In the right panel, antigen specificity was not considered, and only the numbers of total IgA- or IgG-secreting cells were counted. (D) Correlations between the number of gut ASCs making antigen-specific IgA and the level of antigen-specific IgA in serum. EC 50 values represent the serum dilution, which gives half-maximal response in ELISA. The right panel shows the ratio between TG2- and DGP-specific gut ASCs (x axis) and the corresponding ratio between serum antibody levels (y axis). Horizontal lines indicate means, and differences between groups were analyzed using repeated-measures one-way ANOVA. ∗ p
    Figure Legend Snippet: Detection of Antigen-Specific Serum Antibodies and Gut PCs (A) Serum antibody reactivity against TG2 or DGP in patients with untreated celiac disease (UCD, n = 12) or control subjects (n = 12) as determined by ELISA. Sera were diluted 1:200 and added to biotinylated TG2 or biotinylated DGP (biotin-GSGSGS-PLQPEQPFP, harboring an immunodominant gliadin epitope) ( Schwertz et al., 2004 , Steinsbø et al., 2014 ) immobilized on streptavidin followed by detection of bound IgA or IgG using isotype-specific goat anti-human antibodies. The dynamic range of the assay was comparable for detection of the different antibodies. Open symbols indicate subjects from whom serum antibodies were purified for further characterization. The same symbols are used throughout this study. (B) Correlation between the levels of serum IgA and IgG against each of the two antigens in celiac disease patients (n = 20). Reactivity was measured by ELISA, and the signals for each antigen are given relative to the IgA signal obtained with a reference serum sample (UCD1283). (C) ELISPOT detection of total or antigen-specific antibody-secreting cells (ASCs) producing IgA or IgG in gut biopsy specimens obtained from celiac disease patients (n = 8). Cell suspensions were added to coated NeutrAvidin alone or NeutrAvidin associated with biotinylated TG2 or DGP as indicated. In the right panel, antigen specificity was not considered, and only the numbers of total IgA- or IgG-secreting cells were counted. (D) Correlations between the number of gut ASCs making antigen-specific IgA and the level of antigen-specific IgA in serum. EC 50 values represent the serum dilution, which gives half-maximal response in ELISA. The right panel shows the ratio between TG2- and DGP-specific gut ASCs (x axis) and the corresponding ratio between serum antibody levels (y axis). Horizontal lines indicate means, and differences between groups were analyzed using repeated-measures one-way ANOVA. ∗ p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Purification, Enzyme-linked Immunospot

    51) Product Images from "Power Stroke Angular Velocity Profiles of Archaeal A-ATP Synthase Versus Thermophilic and Mesophilic F-ATP Synthase Molecular Motors *"

    Article Title: Power Stroke Angular Velocity Profiles of Archaeal A-ATP Synthase Versus Thermophilic and Mesophilic F-ATP Synthase Molecular Motors *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.745240

    Single molecule assay of rotating protein-gold nanorod complexes. A, schematic model of the microscope setup with a protein-gold nanorod complex. The Mm A 3 B 3 DF complex (subunits A, B, D, and F in orange, green, yellow, and purple, respectively) is attached via its His 8 tags in subunit A to a coverslip, although a neutravidin-coated nanorod is attached to the biotinylated cysteine ( red ) in subunit D. Gold-nanorods were illuminated by a dark-field condenser. Red scattered polarized light was recorded by an APD after passing through a polarizer and a bandpass filter to block shorter wavelengths of light. B, consecutive histograms of light intensity scattered from a nanorod attached to a rotating Mm A 3 B 3 DF complex collected at 1 kHz as a function of the polarization angle during ATPase-powered rotation. C, SDS-9% polyacrylamide gel of purified recombinant Gs α 3 β 3 γ and molecular mass markers. D, consecutive histograms of light intensity scattered from a nanorod attached to a rotating Gs α 3 β 3 γ complex collected at 1 kHz as a function of the polarization angle during ATPase-powered rotation. The approximate courses of the three sinusoidal curves that resulted from the three catalytic dwells are indicated in gray .
    Figure Legend Snippet: Single molecule assay of rotating protein-gold nanorod complexes. A, schematic model of the microscope setup with a protein-gold nanorod complex. The Mm A 3 B 3 DF complex (subunits A, B, D, and F in orange, green, yellow, and purple, respectively) is attached via its His 8 tags in subunit A to a coverslip, although a neutravidin-coated nanorod is attached to the biotinylated cysteine ( red ) in subunit D. Gold-nanorods were illuminated by a dark-field condenser. Red scattered polarized light was recorded by an APD after passing through a polarizer and a bandpass filter to block shorter wavelengths of light. B, consecutive histograms of light intensity scattered from a nanorod attached to a rotating Mm A 3 B 3 DF complex collected at 1 kHz as a function of the polarization angle during ATPase-powered rotation. C, SDS-9% polyacrylamide gel of purified recombinant Gs α 3 β 3 γ and molecular mass markers. D, consecutive histograms of light intensity scattered from a nanorod attached to a rotating Gs α 3 β 3 γ complex collected at 1 kHz as a function of the polarization angle during ATPase-powered rotation. The approximate courses of the three sinusoidal curves that resulted from the three catalytic dwells are indicated in gray .

    Techniques Used: Microscopy, Blocking Assay, Purification, Recombinant

    52) Product Images from "Fission yeast myosin Myo2 is down-regulated in actin affinity by light chain phosphorylation"

    Article Title: Fission yeast myosin Myo2 is down-regulated in actin affinity by light chain phosphorylation

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1703161114

    RLC phosphorylation reduces the number of Myo2 heads bound to actin filaments. Panels show maximum projections of representative fields (128 μm × 128 μm × 50 s) at different KCl concentrations ( A – C ). Phosphorylated Myo2 is shown on the Left (red) and unphosphorylated is shown on the Right ) are shown as averages of multiple fields (N, number of fields). Myo2 was attached to neutravidin-coated coverslips through the C-terminal biotin tag. Conditions were as follows: 30 °C, 25 mM imidazole, pH 7.4, KCl as indicated, 4 mM MgCl 2 , 1 mM EGTA, 1 mM ATP, and 10 mM DTT. No methylcellulose was used to allow the diffusion of actin away from the surface. When indicated, Tpm was added to a final concentration of 2 μM. Data are from two independent preparations of Myo2 (N-FLAG-C-Biotin). Error, ± SD.
    Figure Legend Snippet: RLC phosphorylation reduces the number of Myo2 heads bound to actin filaments. Panels show maximum projections of representative fields (128 μm × 128 μm × 50 s) at different KCl concentrations ( A – C ). Phosphorylated Myo2 is shown on the Left (red) and unphosphorylated is shown on the Right ) are shown as averages of multiple fields (N, number of fields). Myo2 was attached to neutravidin-coated coverslips through the C-terminal biotin tag. Conditions were as follows: 30 °C, 25 mM imidazole, pH 7.4, KCl as indicated, 4 mM MgCl 2 , 1 mM EGTA, 1 mM ATP, and 10 mM DTT. No methylcellulose was used to allow the diffusion of actin away from the surface. When indicated, Tpm was added to a final concentration of 2 μM. Data are from two independent preparations of Myo2 (N-FLAG-C-Biotin). Error, ± SD.

    Techniques Used: Diffusion-based Assay, Concentration Assay

    Effects of RLC phosphorylation on ATPase activity and motility. ( A ) Steady-state ATPase of unphosphorylated (blue) or phosphorylated (red) Myo2 in the absence or presence of tropomyosin (Tpm). Circles and squares represent independent preparations of N-FLAG-Myo2-C-Biotin. Diamonds represent N-FLAG-Myo2. Conditions were as follows: 30 °C, 10 mM imidazole, pH 7.0, 50 mM NaCl, 1 mM MgCl 2 , 1 mM ATP, and 2 mM DTT. Tpm was added at a 2:1 Actin-Tpm molar ratio. ( B ) In vitro motility speeds of unphosphorylated or phosphorylated Myo2, in the absence or presence of Tpm. Speeds represent motility data from two experimental repeats conducted in parallel with two independent preparations of N-FLAG-Myo2-C-Biotin (n, number of moving filaments). To ensure that all heads were available for interaction with actin, Myo2 was attached to neutravidin-coated coverslips by a biotin tag at its C terminus. Conditions were as follows: 30 °C, 0.5% methylcellulose, 25 mM imidazole, pH 7.4, 50 mM KCl, 4 mM MgCl 2 , 1 mM EGTA, 1 mM ATP, and 10 mM DTT. When indicated, Tpm was added to a final concentration of 2 μM.
    Figure Legend Snippet: Effects of RLC phosphorylation on ATPase activity and motility. ( A ) Steady-state ATPase of unphosphorylated (blue) or phosphorylated (red) Myo2 in the absence or presence of tropomyosin (Tpm). Circles and squares represent independent preparations of N-FLAG-Myo2-C-Biotin. Diamonds represent N-FLAG-Myo2. Conditions were as follows: 30 °C, 10 mM imidazole, pH 7.0, 50 mM NaCl, 1 mM MgCl 2 , 1 mM ATP, and 2 mM DTT. Tpm was added at a 2:1 Actin-Tpm molar ratio. ( B ) In vitro motility speeds of unphosphorylated or phosphorylated Myo2, in the absence or presence of Tpm. Speeds represent motility data from two experimental repeats conducted in parallel with two independent preparations of N-FLAG-Myo2-C-Biotin (n, number of moving filaments). To ensure that all heads were available for interaction with actin, Myo2 was attached to neutravidin-coated coverslips by a biotin tag at its C terminus. Conditions were as follows: 30 °C, 0.5% methylcellulose, 25 mM imidazole, pH 7.4, 50 mM KCl, 4 mM MgCl 2 , 1 mM EGTA, 1 mM ATP, and 10 mM DTT. When indicated, Tpm was added to a final concentration of 2 μM.

    Techniques Used: Activity Assay, In Vitro, Concentration Assay

    53) Product Images from "Biochemical and Genetic Characterization of the Enterococcus faecalis Oxaloacetate Decarboxylase Complex"

    Article Title: Biochemical and Genetic Characterization of the Enterococcus faecalis Oxaloacetate Decarboxylase Complex

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.03980-12

    In vitro analysis of Ef -OAD stability as a function of pH. E. faecalis cell extracts were first incubated in batch with a neutravidin resin. After the binding step, the resin was washed (W; lane 3) with 1 ml 150 mM citrate, pH 7.0, and eluted (lanes 4
    Figure Legend Snippet: In vitro analysis of Ef -OAD stability as a function of pH. E. faecalis cell extracts were first incubated in batch with a neutravidin resin. After the binding step, the resin was washed (W; lane 3) with 1 ml 150 mM citrate, pH 7.0, and eluted (lanes 4

    Techniques Used: In Vitro, Incubation, Binding Assay

    54) Product Images from "Pre-Targeting and Direct Immunotargeting of Liposomal Drug Carriers to Ovarian Carcinoma"

    Article Title: Pre-Targeting and Direct Immunotargeting of Liposomal Drug Carriers to Ovarian Carcinoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0041410

    SPECT-CT imaging. SPECT-CT images of pre-targeted and non-targeted 99m Tc-liposomes 4 h (A–B) and 24 h (C–D) after injection of liposomes, administrated either i.v. (A, C ) or i.p. (B, D). Neutravidin-cetuximab was injected to pre-targeted groups and PBS to non-targeted groups i.p. 24 h before liposome injections. Tumors are marked with white circles on the figures. Minimum and maximum values of intensity were adjusted to the same scale for 4 h images and for 24 h images, respectively.
    Figure Legend Snippet: SPECT-CT imaging. SPECT-CT images of pre-targeted and non-targeted 99m Tc-liposomes 4 h (A–B) and 24 h (C–D) after injection of liposomes, administrated either i.v. (A, C ) or i.p. (B, D). Neutravidin-cetuximab was injected to pre-targeted groups and PBS to non-targeted groups i.p. 24 h before liposome injections. Tumors are marked with white circles on the figures. Minimum and maximum values of intensity were adjusted to the same scale for 4 h images and for 24 h images, respectively.

    Techniques Used: Single Photon Emission Computed Tomography, Imaging, Injection

    Flow cytometric analysis of cellular affinity. Directly targeted (black), pre-targeted (red) and non-targeted (blue) fluorescein-labeled liposomes were incubated with SKOV-3 (A) and SKOV3.ip1 (B–C) cells. In the pre-targeting group, the cells were incubated with neutravidin-cetuximab for 4 h, washed and incubated with biotin-liposomes for 2 h (A–B) or 4 h (C). In the other groups, the cells were incubated with the liposomes for 2 h (A–B) or 4 h (C). The green line is representing the background fluorescence of untreated cells.
    Figure Legend Snippet: Flow cytometric analysis of cellular affinity. Directly targeted (black), pre-targeted (red) and non-targeted (blue) fluorescein-labeled liposomes were incubated with SKOV-3 (A) and SKOV3.ip1 (B–C) cells. In the pre-targeting group, the cells were incubated with neutravidin-cetuximab for 4 h, washed and incubated with biotin-liposomes for 2 h (A–B) or 4 h (C). In the other groups, the cells were incubated with the liposomes for 2 h (A–B) or 4 h (C). The green line is representing the background fluorescence of untreated cells.

    Techniques Used: Flow Cytometry, Labeling, Incubation, Fluorescence

    Biodistribution of pre-targeted and non-targeted liposomes in mice bearing i.p. SKOV3.ip1 xenografts. Either neutravidin-cetuximab, at a dose of 20 µg of antibody, or PBS was injected i.p. to the mice. 99m Tc-labeled biotin-liposomes were injected 24 h later i.v. (A) or i.p. (B). Radioactivity of the indicated tissues was determined 24 h from liposome injections. The results are expressed as %ID/g tissue ± SD for pre-targeted liposomes and for non-targeted liposomes ( n = 3–4).
    Figure Legend Snippet: Biodistribution of pre-targeted and non-targeted liposomes in mice bearing i.p. SKOV3.ip1 xenografts. Either neutravidin-cetuximab, at a dose of 20 µg of antibody, or PBS was injected i.p. to the mice. 99m Tc-labeled biotin-liposomes were injected 24 h later i.v. (A) or i.p. (B). Radioactivity of the indicated tissues was determined 24 h from liposome injections. The results are expressed as %ID/g tissue ± SD for pre-targeted liposomes and for non-targeted liposomes ( n = 3–4).

    Techniques Used: Mouse Assay, Injection, Labeling, Radioactivity

    55) Product Images from "Cell Adhesion Assays: Fabrication of an E-cadherin Substratum and Isolation of Lateral and Basal Membrane Patches"

    Article Title: Cell Adhesion Assays: Fabrication of an E-cadherin Substratum and Isolation of Lateral and Basal Membrane Patches

    Journal: Methods in Molecular Biology (Clifton, N.j.)

    doi:

    Fabrication of E-cadherin:Fc substratum. (A) Scheme of chemical assembly of E-cadherin:Fc on a cover slip. A glass cover slip is silianized with a long-chain silane containing an amine, to which sulfo-NHS-biotin is linked. NeutrAvidin and then biotinylated protein A are added sequentially, followed by purified E-cadherin:Fc. E-cadherin on the surface of cells binds to the correctly oriented E-cadherin:Fc substratum. (B) 35 S-methionine/cysteine-labeled E-cadherin:Fc was purified from cells incubated with 35 S-methionine/cysteine as described in Subheading 3.1.3. and used in a dilution series to determine the saturation density of E-cadherin:Fc on the substratum as approx 50,000 molecules/μm 2 .
    Figure Legend Snippet: Fabrication of E-cadherin:Fc substratum. (A) Scheme of chemical assembly of E-cadherin:Fc on a cover slip. A glass cover slip is silianized with a long-chain silane containing an amine, to which sulfo-NHS-biotin is linked. NeutrAvidin and then biotinylated protein A are added sequentially, followed by purified E-cadherin:Fc. E-cadherin on the surface of cells binds to the correctly oriented E-cadherin:Fc substratum. (B) 35 S-methionine/cysteine-labeled E-cadherin:Fc was purified from cells incubated with 35 S-methionine/cysteine as described in Subheading 3.1.3. and used in a dilution series to determine the saturation density of E-cadherin:Fc on the substratum as approx 50,000 molecules/μm 2 .

    Techniques Used: Purification, Labeling, Incubation

    56) Product Images from "A Small Molecule That Inhibits the Interaction of Paxillin and ?4 Integrin Inhibits Accumulation of Mononuclear Leukocytes at a Site of Inflammation *"

    Article Title: A Small Molecule That Inhibits the Interaction of Paxillin and ?4 Integrin Inhibits Accumulation of Mononuclear Leukocytes at a Site of Inflammation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.066993

    Effect of compound 6-B345TTQ and 6-B234TTQ on the binding of leupaxin to α4 integrin. A , biotinylated α4 tail immobilized to neutravidin-coated ELISA plates and incubated with 10 μg/ml leupaxin in the presence of increasing concentrations
    Figure Legend Snippet: Effect of compound 6-B345TTQ and 6-B234TTQ on the binding of leupaxin to α4 integrin. A , biotinylated α4 tail immobilized to neutravidin-coated ELISA plates and incubated with 10 μg/ml leupaxin in the presence of increasing concentrations

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation

    Effect of compound 6-B345TTQ on different paxillin/leupaxin interactions. A , inhibition of binding to α4. Biotinylated α4 tail protein was immobilized to neutravidin-coated ELISA plates and incubated with 10 μg/ml leupaxin or paxillin
    Figure Legend Snippet: Effect of compound 6-B345TTQ on different paxillin/leupaxin interactions. A , inhibition of binding to α4. Biotinylated α4 tail protein was immobilized to neutravidin-coated ELISA plates and incubated with 10 μg/ml leupaxin or paxillin

    Techniques Used: Inhibition, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation

    6-B345TTQ acts as a competitive inhibitor. Biotinylated recombinant α4 tails were immobilized on neutravidin-coated 96-well plates. Bound proteins were incubated with increasing concentrations of HA-tagged recombinant leupaxin in the presence
    Figure Legend Snippet: 6-B345TTQ acts as a competitive inhibitor. Biotinylated recombinant α4 tails were immobilized on neutravidin-coated 96-well plates. Bound proteins were incubated with increasing concentrations of HA-tagged recombinant leupaxin in the presence

    Techniques Used: Recombinant, Incubation

    57) Product Images from "Automated selection of aptamers against protein targets translated in vitro: from gene to aptamer"

    Article Title: Automated selection of aptamers against protein targets translated in vitro: from gene to aptamer

    Journal: Nucleic Acids Research

    doi:

    Detection of in vitro expressed and biotinylated products. Four constructs were investigated for their ability to bind to neutravidin-coated microwells after in vitro expression and biotinylation: β-lactamase (amp), chloramphenicol acetyltransferase (cat), aminoglycoside phosphotransferase (neo) and maltose-binding protein (mbp). Purified, translated and biotinylated products were serially diluted and probed with their respective antisera and again with anti-rabbit IgG–HRP. Signal intensity is measured after incubation of the bound complexes with o -phenylenediamine at 492 nm.
    Figure Legend Snippet: Detection of in vitro expressed and biotinylated products. Four constructs were investigated for their ability to bind to neutravidin-coated microwells after in vitro expression and biotinylation: β-lactamase (amp), chloramphenicol acetyltransferase (cat), aminoglycoside phosphotransferase (neo) and maltose-binding protein (mbp). Purified, translated and biotinylated products were serially diluted and probed with their respective antisera and again with anti-rabbit IgG–HRP. Signal intensity is measured after incubation of the bound complexes with o -phenylenediamine at 492 nm.

    Techniques Used: In Vitro, Construct, Expressing, Binding Assay, Purification, Incubation

    58) Product Images from "Cdc42-dependent modulation of rigidity sensing and cell spreading in tumor repopulating cells"

    Article Title: Cdc42-dependent modulation of rigidity sensing and cell spreading in tumor repopulating cells

    Journal: Biochemical and biophysical research communications

    doi: 10.1016/j.bbrc.2018.04.085

    TRCs do not spread on any TGT surfaces. a , A schematic of a cell on a TGT surface. TGTs with varying T tol ranging from 12 to 56 pN were immobilized on biotinylated BSA passivated glass surfaces via biotin-neutravidin interactions. Biotinylated cyclic-RGDfK peptide, immobilized directly on the surface, was represented as > 100 pN. b , TRCs adhere to surfaces with T tol ≥ 43 pN. Interestingly, TRCs do not spread on any TGT surfaces. c , Projected cell area of TRCs (n=33, 33, 38, 35 for 43 pN, 50 pN, 56 pN, and > 100 pN respectively) are presented in a box-and-whisker plot showing no significant changes across any TGT surfaces (p values are > 0.09, 0.07 and 0.99 for 43 pN and 50 pN, 50 pN and 56 pN, and 56 pN and > 100 pN, respectively). d , A box-and-whisker plot shows a dimensionless parameter-CSI of cells on varying T tol surfaces. No significant changes in CSI values were observed across any TGT surfaces (p values are > 0.78, 0.47, 0.29 for 43 pN and 50 pN, 50 pN and 56 pN, and 56 pN and > 100 pN, respectively).
    Figure Legend Snippet: TRCs do not spread on any TGT surfaces. a , A schematic of a cell on a TGT surface. TGTs with varying T tol ranging from 12 to 56 pN were immobilized on biotinylated BSA passivated glass surfaces via biotin-neutravidin interactions. Biotinylated cyclic-RGDfK peptide, immobilized directly on the surface, was represented as > 100 pN. b , TRCs adhere to surfaces with T tol ≥ 43 pN. Interestingly, TRCs do not spread on any TGT surfaces. c , Projected cell area of TRCs (n=33, 33, 38, 35 for 43 pN, 50 pN, 56 pN, and > 100 pN respectively) are presented in a box-and-whisker plot showing no significant changes across any TGT surfaces (p values are > 0.09, 0.07 and 0.99 for 43 pN and 50 pN, 50 pN and 56 pN, and 56 pN and > 100 pN, respectively). d , A box-and-whisker plot shows a dimensionless parameter-CSI of cells on varying T tol surfaces. No significant changes in CSI values were observed across any TGT surfaces (p values are > 0.78, 0.47, 0.29 for 43 pN and 50 pN, 50 pN and 56 pN, and 56 pN and > 100 pN, respectively).

    Techniques Used: Whisker Assay

    59) Product Images from "Poleward transport of Eg5 by dynein-dynactin in Xenopus laevis egg extract spindles"

    Article Title: Poleward transport of Eg5 by dynein-dynactin in Xenopus laevis egg extract spindles

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200801125

    Microtubule pair sliding driven by purified Eg5 in buffer. (a) Schematic of Eg5 within a pair of antiparallel microtubules. The red microtubule is biotinylated and immobilized to a chemically functionalized glass surface via biotin–neutravidin links. The green microtubule is not biotinylated and mobile. (b) Fluorescence microscopy images of Alexa 568 microtubule pairs formed with Eg5-paGFP. Dimly labeled microtubules immobilized on a biotin-PEG glass surface supporting Eg5-mediated binding of brightly labeled microtubules (see Materials and methods). (c) Time series of confocal fluorescence microscopy images of a Cy5-labeled microtubule (green) moved by Eg5 along an immobilized Alexa 568–labeled microtubule (red). Arrows indicate the initial position of the Cy5-labeled microtubule. The bottom panel shows a kymograph of a line along the microtubule pair during the first 5 min of the time series. (d) Histograms showing distributions for speeds of microtubule sliding driven by Eg5 and Eg5-paGFP.
    Figure Legend Snippet: Microtubule pair sliding driven by purified Eg5 in buffer. (a) Schematic of Eg5 within a pair of antiparallel microtubules. The red microtubule is biotinylated and immobilized to a chemically functionalized glass surface via biotin–neutravidin links. The green microtubule is not biotinylated and mobile. (b) Fluorescence microscopy images of Alexa 568 microtubule pairs formed with Eg5-paGFP. Dimly labeled microtubules immobilized on a biotin-PEG glass surface supporting Eg5-mediated binding of brightly labeled microtubules (see Materials and methods). (c) Time series of confocal fluorescence microscopy images of a Cy5-labeled microtubule (green) moved by Eg5 along an immobilized Alexa 568–labeled microtubule (red). Arrows indicate the initial position of the Cy5-labeled microtubule. The bottom panel shows a kymograph of a line along the microtubule pair during the first 5 min of the time series. (d) Histograms showing distributions for speeds of microtubule sliding driven by Eg5 and Eg5-paGFP.

    Techniques Used: Purification, Fluorescence, Microscopy, Labeling, Binding Assay

    60) Product Images from "Generating Recombinant Antibodies against Putative Biomarkers of Retinal Injury"

    Article Title: Generating Recombinant Antibodies against Putative Biomarkers of Retinal Injury

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0124492

    ELISA of scFv-displayed virions against the GBB5 peptide. ScFv antibodies, raised against the GBB5 peptide, are displayed on phage particles and used to probe peptides, which are captured on microtiter plate well surface. The phage-displayed scFvs are named according to the well location of the 96-well screen in which they were originally found. Specificity is determined by the intensity of the absorbance signal for the target peptide (GBB5), compared to the background NeutrAvidin (NA), Streptavidin (SA), Phosphate Buffered Saline (PBS) coated wells or unrelated peptides, CACNA1F and CNGA3.
    Figure Legend Snippet: ELISA of scFv-displayed virions against the GBB5 peptide. ScFv antibodies, raised against the GBB5 peptide, are displayed on phage particles and used to probe peptides, which are captured on microtiter plate well surface. The phage-displayed scFvs are named according to the well location of the 96-well screen in which they were originally found. Specificity is determined by the intensity of the absorbance signal for the target peptide (GBB5), compared to the background NeutrAvidin (NA), Streptavidin (SA), Phosphate Buffered Saline (PBS) coated wells or unrelated peptides, CACNA1F and CNGA3.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Comparison of scFv and the Fc format of the anti-GBB5-H9. (A) Target peptides are immobilized on a NeutrAvidin-coated microtiter plate at 25 nM and assayed with 30 nM of monomeric E . coli expressed anti-GBB5 H9 scFv or 10 nM of the HEK-293 expressed bivalent Fc format. The secondary antibody anti-FLAG-HRP is added and subsequent chromogenic reagent. Absorbance is recorded at 405 nm. (B) The GBB5-MBP fusion protein (46 kDa) is detected on PVDF membrane using the anti-GBB5-H9 scFv or Fc format of the H9 clone. Amount loaded is in μg and decreases from left to right. The negative control included is the RGS9-MBP fusion protein at 2μg.
    Figure Legend Snippet: Comparison of scFv and the Fc format of the anti-GBB5-H9. (A) Target peptides are immobilized on a NeutrAvidin-coated microtiter plate at 25 nM and assayed with 30 nM of monomeric E . coli expressed anti-GBB5 H9 scFv or 10 nM of the HEK-293 expressed bivalent Fc format. The secondary antibody anti-FLAG-HRP is added and subsequent chromogenic reagent. Absorbance is recorded at 405 nm. (B) The GBB5-MBP fusion protein (46 kDa) is detected on PVDF membrane using the anti-GBB5-H9 scFv or Fc format of the H9 clone. Amount loaded is in μg and decreases from left to right. The negative control included is the RGS9-MBP fusion protein at 2μg.

    Techniques Used: Negative Control

    61) Product Images from "Rifampicin-Independent Interactions between the Pregnane X Receptor Ligand Binding Domain and Peptide Fragments of Coactivator and Corepressor Proteins"

    Article Title: Rifampicin-Independent Interactions between the Pregnane X Receptor Ligand Binding Domain and Peptide Fragments of Coactivator and Corepressor Proteins

    Journal: Biochemistry

    doi: 10.1021/bi2011674

    Specificity of PXR-LBD immobilization. The surface-associated fluorescence of F-PXR-LBD bound to immobilized NeutrAvidin was measured, after surfaces had been treated in the presence (▼) and absence (●) of 100 μ M D-biotin. The
    Figure Legend Snippet: Specificity of PXR-LBD immobilization. The surface-associated fluorescence of F-PXR-LBD bound to immobilized NeutrAvidin was measured, after surfaces had been treated in the presence (▼) and absence (●) of 100 μ M D-biotin. The

    Techniques Used: Fluorescence

    62) Product Images from "Facilitated Preparation of Bioconjugatable Zwitterionic Quantum Dots Using Dual-Lipid Encapsulation"

    Article Title: Facilitated Preparation of Bioconjugatable Zwitterionic Quantum Dots Using Dual-Lipid Encapsulation

    Journal: Journal of colloid and interface science

    doi: 10.1016/j.jcis.2014.09.020

    (a) Illustration of Neutravidin-biotin assay using biotinylated magnetic microbeads and Neutravidin conjugated ZW-QDs; and (b) The photoluminescence responses to biotinylated magnetic microbeads after incubation with non-conjugated and Neutravidin conjugated
    Figure Legend Snippet: (a) Illustration of Neutravidin-biotin assay using biotinylated magnetic microbeads and Neutravidin conjugated ZW-QDs; and (b) The photoluminescence responses to biotinylated magnetic microbeads after incubation with non-conjugated and Neutravidin conjugated

    Techniques Used: Incubation

    63) Product Images from "Nucleotide Discrimination with DNA Immobilized in the MspA Nanopore"

    Article Title: Nucleotide Discrimination with DNA Immobilized in the MspA Nanopore

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025723

    Single heteromeric substitutions in homopolymer ssDNA. Peak values and half width half height values, represented as error bars, of fitted Gaussians of mean residual ionic currents are shown for experiments with a single heteromeric substitution in homopolymer ssDNA. For comparison, the mean residual ionic currents for relevant homopolymer strands are shown as dashed lines. Gaussian curves are included to aid the eye. (A) DNA strands containing a single cytosine (dC) substituted at nucleotide position X from the NeutrAvidin anchor in an otherwise poly-dA strand. Large error bars (X = 13) indicate a wide distribution and the two points at X = 13 and X = 16 indicate two current levels for that substitution. The residual current differs most from that of poly-dA (black dashed line), most resembling that of poly-dC (red dashed line) at X = 14 and 15. (B) DNA strands containing a single cytosine (dA) substituted at nucleotide position X from the NeutrAvidin anchor in an otherwise poly-dC (red) or poly-dT (green) strand. A single dA substitution in homopolymer poly-dC yields a residual current similar to that of poly-dC (red dashed). For a single dA substitution in homopolymer poly-dT, the current deviates most from that of poly-dT (green dashed) towards the level for poly-dA (black-dashed) at X = 14 and 15.
    Figure Legend Snippet: Single heteromeric substitutions in homopolymer ssDNA. Peak values and half width half height values, represented as error bars, of fitted Gaussians of mean residual ionic currents are shown for experiments with a single heteromeric substitution in homopolymer ssDNA. For comparison, the mean residual ionic currents for relevant homopolymer strands are shown as dashed lines. Gaussian curves are included to aid the eye. (A) DNA strands containing a single cytosine (dC) substituted at nucleotide position X from the NeutrAvidin anchor in an otherwise poly-dA strand. Large error bars (X = 13) indicate a wide distribution and the two points at X = 13 and X = 16 indicate two current levels for that substitution. The residual current differs most from that of poly-dA (black dashed line), most resembling that of poly-dC (red dashed line) at X = 14 and 15. (B) DNA strands containing a single cytosine (dA) substituted at nucleotide position X from the NeutrAvidin anchor in an otherwise poly-dC (red) or poly-dT (green) strand. A single dA substitution in homopolymer poly-dC yields a residual current similar to that of poly-dC (red dashed). For a single dA substitution in homopolymer poly-dT, the current deviates most from that of poly-dT (green dashed) towards the level for poly-dA (black-dashed) at X = 14 and 15.

    Techniques Used:

    Methylated cytosine. Mean residual ionic currents and the fitted Gaussian curve are shown for ssDNA of homopolymer cytosine (‘poly-dC’, red) and a strand with 4 methylated cytosines (‘poly 4 -mC’, black) substituted at the 13 th –16 th nucleotides from the NeutrAvidin anchor in an otherwise homopolymer cytosine strand. Experiments were conducted on three pores with poly 4 -mC and poly-dC added sequentially to each pore. The peaks of the Gaussians are separated by 1.1 pA and the curves overlap by ∼2% of the area of the two Gaussians.
    Figure Legend Snippet: Methylated cytosine. Mean residual ionic currents and the fitted Gaussian curve are shown for ssDNA of homopolymer cytosine (‘poly-dC’, red) and a strand with 4 methylated cytosines (‘poly 4 -mC’, black) substituted at the 13 th –16 th nucleotides from the NeutrAvidin anchor in an otherwise homopolymer cytosine strand. Experiments were conducted on three pores with poly 4 -mC and poly-dC added sequentially to each pore. The peaks of the Gaussians are separated by 1.1 pA and the curves overlap by ∼2% of the area of the two Gaussians.

    Techniques Used: Methylation

    Schematic diagram. Schematic diagram of MspA (blue) set up in a lipid bilayer (grey). Single stranded DNA (ssDNA) was attached to a bulky NeutrAvidin molecule (green) using a biotin linker (black). A specific nucleotide (red) is designated by it's position, X, from the biotin-NeutrAvidin ‘anchor’. The ssDNA threads through the pore from the cis side of the bilayer until the bulky NeutrAvidin prevents it from further translocation. Residual ion current was recorded as the ssDNA is immobilized within MspA.
    Figure Legend Snippet: Schematic diagram. Schematic diagram of MspA (blue) set up in a lipid bilayer (grey). Single stranded DNA (ssDNA) was attached to a bulky NeutrAvidin molecule (green) using a biotin linker (black). A specific nucleotide (red) is designated by it's position, X, from the biotin-NeutrAvidin ‘anchor’. The ssDNA threads through the pore from the cis side of the bilayer until the bulky NeutrAvidin prevents it from further translocation. Residual ion current was recorded as the ssDNA is immobilized within MspA.

    Techniques Used: Translocation Assay

    SNP histograms (rs889312). A segment of DNA containing SNP rs889312 was bound to NeutrAvidin such that the polymorphism is at the 13–16 th position from the NeutrAvidin anchor (denoted dC X or dA X where X was the position of the SNP). Part of the surrounding sequence is shown with the SNP highlighted in red (see Table S1 for complete sequence). Histograms of mean residual current levels are shown for each variant and SNP location, X = 13–16. The two variations are most clearly resolved for X = 14 and 15. For reference, the mean residual ionic currents for poly-dC (red dashed) and poly-dA (black dashed) are shown.
    Figure Legend Snippet: SNP histograms (rs889312). A segment of DNA containing SNP rs889312 was bound to NeutrAvidin such that the polymorphism is at the 13–16 th position from the NeutrAvidin anchor (denoted dC X or dA X where X was the position of the SNP). Part of the surrounding sequence is shown with the SNP highlighted in red (see Table S1 for complete sequence). Histograms of mean residual current levels are shown for each variant and SNP location, X = 13–16. The two variations are most clearly resolved for X = 14 and 15. For reference, the mean residual ionic currents for poly-dC (red dashed) and poly-dA (black dashed) are shown.

    Techniques Used: Sequencing, Variant Assay

    64) Product Images from "Deciphering the Interactome of Neisseria meningitidis With Human Brain Microvascular Endothelial Cells"

    Article Title: Deciphering the Interactome of Neisseria meningitidis With Human Brain Microvascular Endothelial Cells

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.02294

    Biotinylation of N. meningitidis proteome and confirmation of the presence of biotinylated proteins bound to hBMECs. (A) Lane 1–Protein extract of meningococci prior to biotinylation separated on SDS-PAGE. Lane 2–Biotinylated proteins were incubated with NeutrAvidin capture beads, eluted with 50 mM DTT and separated on SDS-PAGE. (B) Dot blot. S2–Protein extract of hBMECs obtained after incubation with biotinylated proteins of meningococci spotted on the membrane and detected with IRdye®;800 Streptavidin. Negative control–Total protein extract of hBMECs was spotted on the membrane and incubated with IRdye®;800 Streptavidin, Input control–Biotinylated proteins of meningococci were spotted on the membrane and detected with IRdye®;800 Streptavidin.
    Figure Legend Snippet: Biotinylation of N. meningitidis proteome and confirmation of the presence of biotinylated proteins bound to hBMECs. (A) Lane 1–Protein extract of meningococci prior to biotinylation separated on SDS-PAGE. Lane 2–Biotinylated proteins were incubated with NeutrAvidin capture beads, eluted with 50 mM DTT and separated on SDS-PAGE. (B) Dot blot. S2–Protein extract of hBMECs obtained after incubation with biotinylated proteins of meningococci spotted on the membrane and detected with IRdye®;800 Streptavidin. Negative control–Total protein extract of hBMECs was spotted on the membrane and incubated with IRdye®;800 Streptavidin, Input control–Biotinylated proteins of meningococci were spotted on the membrane and detected with IRdye®;800 Streptavidin.

    Techniques Used: SDS Page, Incubation, Dot Blot, Negative Control

    Schematic representation of experimental and bioinformatic workflow performed in this study. Proteins of N. meningitidis were isolated 1 and biotinylated 2 . Biotinylated proteome was incubated with human brain microvascular endothelial cells (hBMECs) 3A . Unbound proteins were washed off 3Band3C and interacting proteins bound to hBMECs were recovered from cell lyste 3D with NeutrAvidin capture beads 4aand4b , eluted with 50 mM DTT 4c and identified by SWATH-MS 5 . Combination of bioinformatic tools was used for analyzing the interactome of N. meningitidis 6 . Surface proteins were grouped for selection of potential ligands. First analysis 6−I (Psortb and Cello) segregated proteins according to subcellular location and intracellular proteins were removed. With the second set of analytic tools 6−II , interactome was classified into the outer (group 1), inner (group 2) and secretory (group 3) proteins by applying algorithms to predict transmembrane helices (TMHMM and HMMtop) and type I and II signal peptides (SignalP and LipoP). Gene ontology (Blast2GO), UniProt and literature review 6−III were performed further on proteins grouped above in 6-II. Bioinformatic pipeline enabled us to select most probable protein candidates that may interact with hBMECs and contribute in pathogenesis 7 . Those protein candidates were overexpressed in E.coli 8 and used for validation with ELISA and immunocytochemistry 9 .
    Figure Legend Snippet: Schematic representation of experimental and bioinformatic workflow performed in this study. Proteins of N. meningitidis were isolated 1 and biotinylated 2 . Biotinylated proteome was incubated with human brain microvascular endothelial cells (hBMECs) 3A . Unbound proteins were washed off 3Band3C and interacting proteins bound to hBMECs were recovered from cell lyste 3D with NeutrAvidin capture beads 4aand4b , eluted with 50 mM DTT 4c and identified by SWATH-MS 5 . Combination of bioinformatic tools was used for analyzing the interactome of N. meningitidis 6 . Surface proteins were grouped for selection of potential ligands. First analysis 6−I (Psortb and Cello) segregated proteins according to subcellular location and intracellular proteins were removed. With the second set of analytic tools 6−II , interactome was classified into the outer (group 1), inner (group 2) and secretory (group 3) proteins by applying algorithms to predict transmembrane helices (TMHMM and HMMtop) and type I and II signal peptides (SignalP and LipoP). Gene ontology (Blast2GO), UniProt and literature review 6−III were performed further on proteins grouped above in 6-II. Bioinformatic pipeline enabled us to select most probable protein candidates that may interact with hBMECs and contribute in pathogenesis 7 . Those protein candidates were overexpressed in E.coli 8 and used for validation with ELISA and immunocytochemistry 9 .

    Techniques Used: Isolation, Incubation, Mass Spectrometry, Selection, Enzyme-linked Immunosorbent Assay, Immunocytochemistry

    65) Product Images from "Comparison of avidin, neutravidin, and streptavidin as nanocarriers for efficient siRNA Delivery"

    Article Title: Comparison of avidin, neutravidin, and streptavidin as nanocarriers for efficient siRNA Delivery

    Journal: Molecular pharmaceutics

    doi: 10.1021/acs.molpharmaceut.6b00933

    Biodistribution of the neutravidin-based siRNA nanocomplex in rats with CC1 4 -induced liver fibrosis. Cy-5 labeled siRNA was used in this study. The rats were sacrificed two hours post tail vein injection, and major organs including the liver, spleen, kidneys, lungs, heart, and muscle were harvested for fluorescence imaging analysis using a Bruker MS FX PRO Imaging System. Fluorescence images of the major organs of four rats per group were presented in (A). (B). Region of interest (ROI) was determined by the Bruker molecular imaging software. Fluorescence intensities with respect to the area under the ROI were plotted for the liver, spleen, kidneys, lungs, heart and muscle. Results are represented as the mean ± SD (n=4; ** P≤0.01).
    Figure Legend Snippet: Biodistribution of the neutravidin-based siRNA nanocomplex in rats with CC1 4 -induced liver fibrosis. Cy-5 labeled siRNA was used in this study. The rats were sacrificed two hours post tail vein injection, and major organs including the liver, spleen, kidneys, lungs, heart, and muscle were harvested for fluorescence imaging analysis using a Bruker MS FX PRO Imaging System. Fluorescence images of the major organs of four rats per group were presented in (A). (B). Region of interest (ROI) was determined by the Bruker molecular imaging software. Fluorescence intensities with respect to the area under the ROI were plotted for the liver, spleen, kidneys, lungs, heart and muscle. Results are represented as the mean ± SD (n=4; ** P≤0.01).

    Techniques Used: Labeling, Injection, Fluorescence, Imaging, Mass Spectrometry, Software

    Characterization of the avidin-, neutravidin-, and streptavidin-based siRNA nanocomplexes. (A) Particle size; (B) Zeta potential; (C) Complexation of biotin-siRNA, biotin-cholesterol, with avidin analogues.
    Figure Legend Snippet: Characterization of the avidin-, neutravidin-, and streptavidin-based siRNA nanocomplexes. (A) Particle size; (B) Zeta potential; (C) Complexation of biotin-siRNA, biotin-cholesterol, with avidin analogues.

    Techniques Used: Avidin-Biotin Assay

    Cellular uptake of the avidin nanocomplex (A), neutravidin nanocomplex (B), and streptavidin nanocomplex (C) in HSC-T6 cells. Alexa Fluor-647 labeled siRNA was encapsulated in nanocomplexes and confocal microscopy was used to monitor the cellular uptake at different time intervals (3, 6, and 24 h).
    Figure Legend Snippet: Cellular uptake of the avidin nanocomplex (A), neutravidin nanocomplex (B), and streptavidin nanocomplex (C) in HSC-T6 cells. Alexa Fluor-647 labeled siRNA was encapsulated in nanocomplexes and confocal microscopy was used to monitor the cellular uptake at different time intervals (3, 6, and 24 h).

    Techniques Used: Avidin-Biotin Assay, Labeling, Confocal Microscopy

    Quantitative cellular uptake study of the avidin-, neutravidin- and streptavidin-based nanocomplexes. Cellular uptake of nanocomplexes encapsulating Alexa Fluor-647 labeled siRNA was quantitated using flow cytometry. (A) Percent of cells that take up the nanocomplex; (B) Fluorescence intensity of the cells at various time points; (C) Histogram Plot for the Alexa Fluor-647 siRNA mediated intensity peak shift by avidin-, neutravidin- and streptavidin-based nanocomplexes at various time points. The negative control gate was set at 10 2 APC-A (shown in black). APC-A is the equivalent fluorescent filter for the Alexa Fluor-647.
    Figure Legend Snippet: Quantitative cellular uptake study of the avidin-, neutravidin- and streptavidin-based nanocomplexes. Cellular uptake of nanocomplexes encapsulating Alexa Fluor-647 labeled siRNA was quantitated using flow cytometry. (A) Percent of cells that take up the nanocomplex; (B) Fluorescence intensity of the cells at various time points; (C) Histogram Plot for the Alexa Fluor-647 siRNA mediated intensity peak shift by avidin-, neutravidin- and streptavidin-based nanocomplexes at various time points. The negative control gate was set at 10 2 APC-A (shown in black). APC-A is the equivalent fluorescent filter for the Alexa Fluor-647.

    Techniques Used: Avidin-Biotin Assay, Labeling, Flow Cytometry, Cytometry, Fluorescence, Negative Control

    (A) Silencing activity of avidin-, neutravidin- and streptavidin-based nanocomplexes (100 nM siRNA) at 6 and 24 h. (B). Silencing activity of avidin-, neutravidin- and streptavidin-based nanocomplexes in the presence of 10% FBS at 6 h. Nanocomplexes formulated with PCBP2 siRNA or scrambled siRNA were incubated with HSC-T6 cells in OptiMEM or DMEM supplemented with 10% FBS. Total RNA was isolated and silencing activity was evaluated by Real Time RT-PCR. Results are represented as the mean±SD (n=3).
    Figure Legend Snippet: (A) Silencing activity of avidin-, neutravidin- and streptavidin-based nanocomplexes (100 nM siRNA) at 6 and 24 h. (B). Silencing activity of avidin-, neutravidin- and streptavidin-based nanocomplexes in the presence of 10% FBS at 6 h. Nanocomplexes formulated with PCBP2 siRNA or scrambled siRNA were incubated with HSC-T6 cells in OptiMEM or DMEM supplemented with 10% FBS. Total RNA was isolated and silencing activity was evaluated by Real Time RT-PCR. Results are represented as the mean±SD (n=3).

    Techniques Used: Activity Assay, Avidin-Biotin Assay, Incubation, Isolation, Quantitative RT-PCR

    Nanocomplex enhances serum stability of siRNA. The avidin-, neutravidin-, and streptavidin-based nanocomplexes were incubated with 50% rat serum for 0, 12 and 24 h. The samples were incubated with 40 μM heparin and 100 mM DTT for 30 min to release free siRNA from the nanocomplex and then analyzed with 2% agarose gel.
    Figure Legend Snippet: Nanocomplex enhances serum stability of siRNA. The avidin-, neutravidin-, and streptavidin-based nanocomplexes were incubated with 50% rat serum for 0, 12 and 24 h. The samples were incubated with 40 μM heparin and 100 mM DTT for 30 min to release free siRNA from the nanocomplex and then analyzed with 2% agarose gel.

    Techniques Used: Avidin-Biotin Assay, Incubation, Agarose Gel Electrophoresis

    Analysis of apoptosis and necrosis by propidium iodide staining and flow cytometry. HSC-T6 cells were treated with the avidin-, neutravidin- and streptavidin-based nanocomplexes (100 nM PCBP2 siRNA) for 24 h and then subjected to the analysis of apoptosis and necrosis. The acquisition data were divided into four quadrants according to the type of fluorescence emitted from the cells: Ql-1 calculates the percent of cells undergoing apoptosis (Annexin V), Q2–1 calculates the percent of cells undergoing late apoptosis or induced necrosis (Annexin V and Propidium Iodide), Q3–1 calculates the percent of cells with no fluorescence and Q4–1 calculates the percent of cells undergoing necrosis induced by the nanocomplex. Results are represented as the mean±SD (n=3).
    Figure Legend Snippet: Analysis of apoptosis and necrosis by propidium iodide staining and flow cytometry. HSC-T6 cells were treated with the avidin-, neutravidin- and streptavidin-based nanocomplexes (100 nM PCBP2 siRNA) for 24 h and then subjected to the analysis of apoptosis and necrosis. The acquisition data were divided into four quadrants according to the type of fluorescence emitted from the cells: Ql-1 calculates the percent of cells undergoing apoptosis (Annexin V), Q2–1 calculates the percent of cells undergoing late apoptosis or induced necrosis (Annexin V and Propidium Iodide), Q3–1 calculates the percent of cells with no fluorescence and Q4–1 calculates the percent of cells undergoing necrosis induced by the nanocomplex. Results are represented as the mean±SD (n=3).

    Techniques Used: Staining, Flow Cytometry, Cytometry, Avidin-Biotin Assay, Fluorescence

    Inflammatory cytokine induction study of nanocomplexes in whole blood. Sprague-Dawley rat whole blood was incubated with the avidin-, neutravidin- and streptavidin-based nanocomplexes at 37°C for 24h (A) and 48h (B). The plasma was collected and quantified for the expression of IFNγ, TNF α and IL6 using ELSA kits. Results are represented as the mean ± SD (n=3; * P ≤ 0.05; ** P ≤ 0.01).
    Figure Legend Snippet: Inflammatory cytokine induction study of nanocomplexes in whole blood. Sprague-Dawley rat whole blood was incubated with the avidin-, neutravidin- and streptavidin-based nanocomplexes at 37°C for 24h (A) and 48h (B). The plasma was collected and quantified for the expression of IFNγ, TNF α and IL6 using ELSA kits. Results are represented as the mean ± SD (n=3; * P ≤ 0.05; ** P ≤ 0.01).

    Techniques Used: Incubation, Avidin-Biotin Assay, Expressing

    Exocytosis study of avidin-, neutravidin-, and streptavidin-based nanocomplexes. HSC-T6 cells were transfected with nanocomplex containing Alexa Fluor 647-siRNA for 6 h, and the medium was replaced with fresh OptiMEM medium. The fresh medium was collected at various time intervals and treated with heparin for 30 min and further analyzed for fluorescence intensity using a Victor × fluorescence plate reader. Results are represented as the mean ± SD (n = 3; * P ≤ 0.05; ** P ≤ 0.01).
    Figure Legend Snippet: Exocytosis study of avidin-, neutravidin-, and streptavidin-based nanocomplexes. HSC-T6 cells were transfected with nanocomplex containing Alexa Fluor 647-siRNA for 6 h, and the medium was replaced with fresh OptiMEM medium. The fresh medium was collected at various time intervals and treated with heparin for 30 min and further analyzed for fluorescence intensity using a Victor × fluorescence plate reader. Results are represented as the mean ± SD (n = 3; * P ≤ 0.05; ** P ≤ 0.01).

    Techniques Used: Avidin-Biotin Assay, Transfection, Fluorescence

    66) Product Images from "Differential substrate-induced signaling through the TonB-dependent transporter BtuB"

    Article Title: Differential substrate-induced signaling through the TonB-dependent transporter BtuB

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1932538100

    Reaction of BMCC with E. coli cells expressing BtuB with Cys substitutions. Strain RK5016 carrying pAG1 plasmid derivatives encoding BtuB variants with the indicated single Cys substitutions between positions 6 and 12 or the wild-type Cys-free protein were exposed to the indicated addition and BMCC (15 μg/ml) as described in Experimental Procedures . Cell extracts were resolved by SDS/PAGE, transferred to nitrocellulose membrane, probed with horseradish peroxidase-labeled neutravidin, and visualized by chemiluminescence. Molecular mass markers are indicated on the left in kilodaltons, and the position of BtuB is indicated on the right. ( A ) Addition of 5 μM CNCbl (lanes marked +). ( B ) Addition of E3R (+).
    Figure Legend Snippet: Reaction of BMCC with E. coli cells expressing BtuB with Cys substitutions. Strain RK5016 carrying pAG1 plasmid derivatives encoding BtuB variants with the indicated single Cys substitutions between positions 6 and 12 or the wild-type Cys-free protein were exposed to the indicated addition and BMCC (15 μg/ml) as described in Experimental Procedures . Cell extracts were resolved by SDS/PAGE, transferred to nitrocellulose membrane, probed with horseradish peroxidase-labeled neutravidin, and visualized by chemiluminescence. Molecular mass markers are indicated on the left in kilodaltons, and the position of BtuB is indicated on the right. ( A ) Addition of 5 μM CNCbl (lanes marked +). ( B ) Addition of E3R (+).

    Techniques Used: Expressing, Plasmid Preparation, SDS Page, Labeling

    67) Product Images from "The archaeal “7 kDa DNA-binding” proteins: extended characterization of an old gifted family"

    Article Title: The archaeal “7 kDa DNA-binding” proteins: extended characterization of an old gifted family

    Journal: Scientific Reports

    doi: 10.1038/srep37274

    Sequence selectivity of proteins for dsDNA. Proteins show similar binding preferences by ELISA. Plates were coated with 1 μg/mL Neutravidin and 2.5 ng per well of biotinylated dsDNA were immobilized. Proteins were added at 200 nM. Binding of proteins to dsDNA was detected with anti-RGS(His)6-HRP antibody conjugate. As the recorded absorbance is proportional to the amount of bound protein, higher values correspond to higher affinities for dsDNA. Results are representative of 3 experiments.
    Figure Legend Snippet: Sequence selectivity of proteins for dsDNA. Proteins show similar binding preferences by ELISA. Plates were coated with 1 μg/mL Neutravidin and 2.5 ng per well of biotinylated dsDNA were immobilized. Proteins were added at 200 nM. Binding of proteins to dsDNA was detected with anti-RGS(His)6-HRP antibody conjugate. As the recorded absorbance is proportional to the amount of bound protein, higher values correspond to higher affinities for dsDNA. Results are representative of 3 experiments.

    Techniques Used: Sequencing, Binding Assay, Enzyme-linked Immunosorbent Assay

    68) Product Images from "Combining Single RNA Sensitive Probes with Subdiffraction-Limited and Live-Cell Imaging Enables the Characterization of Virus Dynamics in Cells"

    Article Title: Combining Single RNA Sensitive Probes with Subdiffraction-Limited and Live-Cell Imaging Enables the Characterization of Virus Dynamics in Cells

    Journal: ACS Nano

    doi: 10.1021/nn405998v

    Characterization of MTRIP-labeled hRSV virions. (A) MTRIP-labeled virions were immobilized on a coverglass, and hRSV proteins N and F were detected via immunofluorescence. A representative field of view and a magnified filamentous virion are shown. The hRSV N (green), hRSV F (blue), and labeled gRNA (red) are shown individually as well as merged. (B) Profile plot of the fluorescence intensity along the length of the filament shown in part A. (C) Dark-field images of gold-streptavidin MTRIP-labeled hRSV and neutravidin MTRIP-labeled hRSV. Samples were silver enhanced prior to imaging. Field of view is shown on the left; boxed region indicates single filament featured in the magnified image to the right. (D) Spectra of light emitted from one pixel in the single-filament image (indicated by arrow) for Au-streptavidin-labeled virus (top) or neutravidin-labeled virus (bottom). (E) Effect of varying MTRIP probe concentration on the number of objects detected on the coverglass from infected cells containing hRSV N, hRSV F, and gRNA (above, Kruskal–Wallis one-way ANOVA, * indicates p
    Figure Legend Snippet: Characterization of MTRIP-labeled hRSV virions. (A) MTRIP-labeled virions were immobilized on a coverglass, and hRSV proteins N and F were detected via immunofluorescence. A representative field of view and a magnified filamentous virion are shown. The hRSV N (green), hRSV F (blue), and labeled gRNA (red) are shown individually as well as merged. (B) Profile plot of the fluorescence intensity along the length of the filament shown in part A. (C) Dark-field images of gold-streptavidin MTRIP-labeled hRSV and neutravidin MTRIP-labeled hRSV. Samples were silver enhanced prior to imaging. Field of view is shown on the left; boxed region indicates single filament featured in the magnified image to the right. (D) Spectra of light emitted from one pixel in the single-filament image (indicated by arrow) for Au-streptavidin-labeled virus (top) or neutravidin-labeled virus (bottom). (E) Effect of varying MTRIP probe concentration on the number of objects detected on the coverglass from infected cells containing hRSV N, hRSV F, and gRNA (above, Kruskal–Wallis one-way ANOVA, * indicates p

    Techniques Used: Labeling, Immunofluorescence, Fluorescence, Imaging, Concentration Assay, Infection

    Illustration of MTRIP delivery and virus isolation methodology. (A) The MTRIP design consists of 2′- O -methyl RNA/DNA oligonucleotides that are internally labeled with fluorophores (red dots with carbon linkers on deoxythymidine nucleotides) and biotinylated on the 5′ end (green dot). These oligonucleotides are tetramerized by mixing them with neutravidin (blue circle). The actual probe sequence is shown. (B) HEp-2 cells were infected with hRSV for 96 h before MTRIP probe delivery. A depiction of a cell is shown on the left, and a viral filament protruding out of the cell membrane is shown on the right. A single intergenic sequence of the hRSV genome is shown just below the filament (the ellipsis on either end of the sequence represents the additional 15kb of the hRSV genome). (C) During probe delivery, streptolysin-O is used to permeablize the cell membrane (step 1), allowing MTRIP probes to diffuse into the cell cytoplasm and bind to the intergenic sites of the hRSV genome via Watson–Crick base-pairing (step 2). (D) Streptolysin-O is removed, allowing the pores in the cell membrane to reseal and the hRSV genome/MTRIP complex to be loaded into both spherical and filamentous virion (step 1). The labeled virions are detached from the cell by scraping and can be aliquoted and frozen for further experiments (step 2).
    Figure Legend Snippet: Illustration of MTRIP delivery and virus isolation methodology. (A) The MTRIP design consists of 2′- O -methyl RNA/DNA oligonucleotides that are internally labeled with fluorophores (red dots with carbon linkers on deoxythymidine nucleotides) and biotinylated on the 5′ end (green dot). These oligonucleotides are tetramerized by mixing them with neutravidin (blue circle). The actual probe sequence is shown. (B) HEp-2 cells were infected with hRSV for 96 h before MTRIP probe delivery. A depiction of a cell is shown on the left, and a viral filament protruding out of the cell membrane is shown on the right. A single intergenic sequence of the hRSV genome is shown just below the filament (the ellipsis on either end of the sequence represents the additional 15kb of the hRSV genome). (C) During probe delivery, streptolysin-O is used to permeablize the cell membrane (step 1), allowing MTRIP probes to diffuse into the cell cytoplasm and bind to the intergenic sites of the hRSV genome via Watson–Crick base-pairing (step 2). (D) Streptolysin-O is removed, allowing the pores in the cell membrane to reseal and the hRSV genome/MTRIP complex to be loaded into both spherical and filamentous virion (step 1). The labeled virions are detached from the cell by scraping and can be aliquoted and frozen for further experiments (step 2).

    Techniques Used: Virus Isolation Assay, Labeling, Sequencing, Infection

    69) Product Images from "Rapid and Selective Screening for Sulfhydryl Analytes in Plasma and Urine using Surface-Enhanced Transmission Mode Desorption Electrospray Ionization Mass Spectrometry"

    Article Title: Rapid and Selective Screening for Sulfhydryl Analytes in Plasma and Urine using Surface-Enhanced Transmission Mode Desorption Electrospray Ionization Mass Spectrometry

    Journal: Analytical chemistry

    doi: 10.1021/ac100242b

    Schematic view of surface-enhanced transmission mode desorption electrospray ionization employing VICAT SH as a thiol capture reagent attached to a neutravidin-coated mesh.
    Figure Legend Snippet: Schematic view of surface-enhanced transmission mode desorption electrospray ionization employing VICAT SH as a thiol capture reagent attached to a neutravidin-coated mesh.

    Techniques Used: Transmission Assay

    70) Product Images from "Daptomycin, a last-resort antibiotic, binds ribosomal protein S19 in humans"

    Article Title: Daptomycin, a last-resort antibiotic, binds ribosomal protein S19 in humans

    Journal: Proteome Science

    doi: 10.1186/s12953-017-0124-2

    The biopanning process begins with biotinylation and immobilization of a small molecule onto a surface coated with neutravidin ( red ). Introduction of a DNA library displaying a small number (1–15) of copies of the encoded protein, is introduced into the well and, non-binding phages removed by washing. Bound phages are eluted and amplified ( E. coli BLT5615) and the process repeated until the library converges on the most avid binding target protein(s). Individual phage plaques are sequenced to determine the identity of the displayed protein
    Figure Legend Snippet: The biopanning process begins with biotinylation and immobilization of a small molecule onto a surface coated with neutravidin ( red ). Introduction of a DNA library displaying a small number (1–15) of copies of the encoded protein, is introduced into the well and, non-binding phages removed by washing. Bound phages are eluted and amplified ( E. coli BLT5615) and the process repeated until the library converges on the most avid binding target protein(s). Individual phage plaques are sequenced to determine the identity of the displayed protein

    Techniques Used: Binding Assay, Amplification

    Agarose gel electrophoresis of phage DNA inserts amplified by PCR from cDNA libraries (colon, colon tumor, breast tumor, liver tumor and lung tumor) after 9–12 rounds of biopanning against B-DAP immobilized on a neutravidin-coated microtiter strip wells
    Figure Legend Snippet: Agarose gel electrophoresis of phage DNA inserts amplified by PCR from cDNA libraries (colon, colon tumor, breast tumor, liver tumor and lung tumor) after 9–12 rounds of biopanning against B-DAP immobilized on a neutravidin-coated microtiter strip wells

    Techniques Used: Agarose Gel Electrophoresis, Amplification, Polymerase Chain Reaction, Stripping Membranes

    On-phage binding study comparing the affinity of the RPS19-displaying phage clone C1 (from liver tumor sublibrary of round-9) for neutravidin-coated plates derivatized with a control compound and a similar plate derivatized with B-DAP
    Figure Legend Snippet: On-phage binding study comparing the affinity of the RPS19-displaying phage clone C1 (from liver tumor sublibrary of round-9) for neutravidin-coated plates derivatized with a control compound and a similar plate derivatized with B-DAP

    Techniques Used: Binding Assay

    Agarose gel electrophoresis of PCR products obtained from normal colon, breast tumor, colon tumor, liver tumor and lung tumor individual plaques after nine rounds of selection with B-DAP immobilized on a neutravidin-coated plate. The DNA inserts, which were amplified using generic T7 primers, were also digested with Hin fI to produce unique DNA fingerprints of each clone. Clones that appeared more than once were sequenced
    Figure Legend Snippet: Agarose gel electrophoresis of PCR products obtained from normal colon, breast tumor, colon tumor, liver tumor and lung tumor individual plaques after nine rounds of selection with B-DAP immobilized on a neutravidin-coated plate. The DNA inserts, which were amplified using generic T7 primers, were also digested with Hin fI to produce unique DNA fingerprints of each clone. Clones that appeared more than once were sequenced

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Selection, Amplification, Clone Assay

    71) Product Images from "SSB Functions as a Sliding Platform that Migrates on DNA via Reptation"

    Article Title: SSB Functions as a Sliding Platform that Migrates on DNA via Reptation

    Journal: Cell

    doi: 10.1016/j.cell.2011.06.036

    Force-induced Unraveling of ssDNA from SSB Measured by Fluorescence-Force Spectroscopy (A) Experimental scheme for force-induced unravelling of ssDNA, (dT) 69+1 , from SSB measured via FRET. One end of the construct was immobilized on a PEG surface via biotin-neutravidin interaction and the other end was linked to a bead held in an optical trap via a Digoxigenin-Anti-digoxigenin interaction. (B) Structural model for an SSB tetramer bound to a 70nt ssDNA (thick orange line) in the fully wrapped (SSB) 65 binding mode, based on an X-ray crystallographic structure of a C-terminal truncated SSB tetramer (SSBΔC) bound to two (dC) 35 ). (C) FRET histograms of the DNA construct at zero force with and without SSB bound. The peak at zero FRET corresponds to DNA molecules with active Cy3 only, and the second major peak corresponds to DNA molecules with both active Cy3 and Cy5. Excess SSB proteins were removed from the solution after incubating at 500 mM NaCl and 1 nM SSB tetramer concentration. The FRET histograms were obtained 1 minute, 2 hours, and 5 hours after the removal of free SSB in solution. (D) Fluorescence-force traces obtained while stretching and relaxing the DNA at the stage-moving speed v of 455 nm s −1 (20 nM SSB in solution) when the maximum force achieved was set to ~ 6 pN (Averaged among ~ 50 cycles from 10 molecules with a bin size of 0.2 pN). (E) The averaged FRET vs. force curves for stretching and relaxing the DNA when the maximum force achieved was set to ~ 6 pN (in 500 mM Na + ) or ~8 pN (in 5 mM Mg 2+ and 100 mM Na + ). (F) Unraveling distance vs. force curves in two ionic conditions fit to straight lines (red lines), D = α ·( F-β ) (when the force F ≥ β ), where α = 1.0 ± 0.03 nm/pN, β = 0.9 ± 0.2 pN for 500 mM Na + , and α = 0.7± 0.02 nm/pN, β = 1.2 ± 0.3 pN for 5 mM Mg 2+ and 100 mM Na + , determined from the fit Error bars are the standard errors. .
    Figure Legend Snippet: Force-induced Unraveling of ssDNA from SSB Measured by Fluorescence-Force Spectroscopy (A) Experimental scheme for force-induced unravelling of ssDNA, (dT) 69+1 , from SSB measured via FRET. One end of the construct was immobilized on a PEG surface via biotin-neutravidin interaction and the other end was linked to a bead held in an optical trap via a Digoxigenin-Anti-digoxigenin interaction. (B) Structural model for an SSB tetramer bound to a 70nt ssDNA (thick orange line) in the fully wrapped (SSB) 65 binding mode, based on an X-ray crystallographic structure of a C-terminal truncated SSB tetramer (SSBΔC) bound to two (dC) 35 ). (C) FRET histograms of the DNA construct at zero force with and without SSB bound. The peak at zero FRET corresponds to DNA molecules with active Cy3 only, and the second major peak corresponds to DNA molecules with both active Cy3 and Cy5. Excess SSB proteins were removed from the solution after incubating at 500 mM NaCl and 1 nM SSB tetramer concentration. The FRET histograms were obtained 1 minute, 2 hours, and 5 hours after the removal of free SSB in solution. (D) Fluorescence-force traces obtained while stretching and relaxing the DNA at the stage-moving speed v of 455 nm s −1 (20 nM SSB in solution) when the maximum force achieved was set to ~ 6 pN (Averaged among ~ 50 cycles from 10 molecules with a bin size of 0.2 pN). (E) The averaged FRET vs. force curves for stretching and relaxing the DNA when the maximum force achieved was set to ~ 6 pN (in 500 mM Na + ) or ~8 pN (in 5 mM Mg 2+ and 100 mM Na + ). (F) Unraveling distance vs. force curves in two ionic conditions fit to straight lines (red lines), D = α ·( F-β ) (when the force F ≥ β ), where α = 1.0 ± 0.03 nm/pN, β = 0.9 ± 0.2 pN for 500 mM Na + , and α = 0.7± 0.02 nm/pN, β = 1.2 ± 0.3 pN for 5 mM Mg 2+ and 100 mM Na + , determined from the fit Error bars are the standard errors. .

    Techniques Used: Fluorescence, Spectroscopy, Construct, Binding Assay, Concentration Assay

    72) Product Images from "Selection for improved subtiligases by phage display"

    Article Title: Selection for improved subtiligases by phage display

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    ( A ) Ligation reaction catalyzed by subtiligase. ( B ) Scheme for selecting active subtiligase mutants on phage by requiring that the enzyme attach a biotin-labeled peptide onto its extended N terminus. The biotin-labeled subtiligase phage are then captured with immobilized neutravidin.
    Figure Legend Snippet: ( A ) Ligation reaction catalyzed by subtiligase. ( B ) Scheme for selecting active subtiligase mutants on phage by requiring that the enzyme attach a biotin-labeled peptide onto its extended N terminus. The biotin-labeled subtiligase phage are then captured with immobilized neutravidin.

    Techniques Used: Ligation, Labeling

    73) Product Images from "Complement Activation by CpG in a Human Whole Blood Loop System: Mechanisms and Immunomodulatory Effects 1"

    Article Title: Complement Activation by CpG in a Human Whole Blood Loop System: Mechanisms and Immunomodulatory Effects 1

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.0902374

    CpG 2006 facilitates AP convertase build-up. QCM-D was used to study the interaction of CpG 2006 with C3b. A , A gold sensor was covered with fibrinogen that was biotinylated in the QCM chamber. Subsequently, NeutrAvidin and biotinylated CpG were added.
    Figure Legend Snippet: CpG 2006 facilitates AP convertase build-up. QCM-D was used to study the interaction of CpG 2006 with C3b. A , A gold sensor was covered with fibrinogen that was biotinylated in the QCM chamber. Subsequently, NeutrAvidin and biotinylated CpG were added.

    Techniques Used: QCM-D

    74) Product Images from "Biosensing for the Environment and Defence: Aqueous Uranyl Detection Using Bacterial Surface Layer Proteins"

    Article Title: Biosensing for the Environment and Defence: Aqueous Uranyl Detection Using Bacterial Surface Layer Proteins

    Journal: Sensors (Basel, Switzerland)

    doi: 10.3390/s100504739

    Schematic showing the two alternate tethering methods for SLP incorporation on to gold surfaces. (A) mSAM incorporation of SLP by MHDA/biotin-caproyl-DPPE mSAM, deposited with a Neutravidin layer that binds to pre-biotinylated SLP. (B) Porous membrane model with molecular linkers of 1.5 nm length binding SLP through a stable permeable membrane as maleimide groups covalently bind to thiols on protein cysteine residues.
    Figure Legend Snippet: Schematic showing the two alternate tethering methods for SLP incorporation on to gold surfaces. (A) mSAM incorporation of SLP by MHDA/biotin-caproyl-DPPE mSAM, deposited with a Neutravidin layer that binds to pre-biotinylated SLP. (B) Porous membrane model with molecular linkers of 1.5 nm length binding SLP through a stable permeable membrane as maleimide groups covalently bind to thiols on protein cysteine residues.

    Techniques Used: Binding Assay

    75) Product Images from "Simultaneous Protein Expression and Modification: An Efficient Approach for Production of Unphosphorylated and Biotinylated Receptor Tyrosine Kinases by Triple Infection in the Baculovirus Expression System"

    Article Title: Simultaneous Protein Expression and Modification: An Efficient Approach for Production of Unphosphorylated and Biotinylated Receptor Tyrosine Kinases by Triple Infection in the Baculovirus Expression System

    Journal: Journal of Biomolecular Techniques : JBT

    doi:

    Functional analysis of purified IGF1R and IR cytoplasmic domains. ( A ) The accessibility of the biotin for immobilization was assessed by binding of untagged and biotinylated AviTag-IGF1R to neutravidin-coated CM5 chips on a Biacore T100. ( B ) The enzyme
    Figure Legend Snippet: Functional analysis of purified IGF1R and IR cytoplasmic domains. ( A ) The accessibility of the biotin for immobilization was assessed by binding of untagged and biotinylated AviTag-IGF1R to neutravidin-coated CM5 chips on a Biacore T100. ( B ) The enzyme

    Techniques Used: Functional Assay, Purification, Binding Assay

    Related Articles

    Surface Biotinylation Assay:

    Article Title: Emerging roles of ARHGAP33 in intracellular trafficking of TrkB and pathophysiology of neuropsychiatric disorders
    Article Snippet: Paragraph title: Surface biotinylation assay ... To collect the surface proteins, cells were lysed with lysis buffer (20 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 0.01% SDS) and biotinylated proteins were precipitated with NeutrAvidin resins (Pierce).

    Nucleic Acid Electrophoresis:

    Article Title: Polyubiquitination of Prolactin Receptor Stimulates Its Internalization, Postinternalization Sorting, and Degradation via the Lysosomal Pathway ▿Polyubiquitination of Prolactin Receptor Stimulates Its Internalization, Postinternalization Sorting, and Degradation via the Lysosomal Pathway ▿ †
    Article Snippet: Lysed cells were transferred to a cold labeled tube, incubated on ice for 20 min, and centrifuged at 15,000 rpm for 15 min. Biotinylated proteins were recovered by incubating with immobilized NeutrAvidin resins (Pierce Inc.) overnight. .. The proteins were washed, boiled in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer, separated by 10% SDS-PAGE, and analyzed by immunoblotting with anti-Flag M2 antibody to detect Flag-tagged PRLr.

    In Vitro:

    Article Title: Emerging roles of ARHGAP33 in intracellular trafficking of TrkB and pathophysiology of neuropsychiatric disorders
    Article Snippet: The surface proteins of hippocampal cultures at 14 days in vitro and HEK293T cells were biotinylated with 1 mg ml−1 sulfo-NHS-SS-biotin (Pierce, IL, USA) for 20 min at 4 °C. .. To collect the surface proteins, cells were lysed with lysis buffer (20 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 0.01% SDS) and biotinylated proteins were precipitated with NeutrAvidin resins (Pierce).

    Protease Inhibitor:

    Article Title: Polyubiquitination of Prolactin Receptor Stimulates Its Internalization, Postinternalization Sorting, and Degradation via the Lysosomal Pathway ▿Polyubiquitination of Prolactin Receptor Stimulates Its Internalization, Postinternalization Sorting, and Degradation via the Lysosomal Pathway ▿ †
    Article Snippet: After two additional washes with ice-cold HBS, cells were lysed in the plate with 60 mM n -octyl β- d -glucopyranoside (Fisher) and 0.1% sodium dodecyl sulfate (SDS) in HBS containing protease inhibitor cocktail. .. Lysed cells were transferred to a cold labeled tube, incubated on ice for 20 min, and centrifuged at 15,000 rpm for 15 min. Biotinylated proteins were recovered by incubating with immobilized NeutrAvidin resins (Pierce Inc.) overnight.

    Article Title: LMTK3 Deficiency Causes Pronounced Locomotor Hyperactivity and Impairs Endocytic Trafficking
    Article Snippet: .. To collect surface proteins, cells were lysed with lysis buffer (20 m m HEPES, pH 7.5, 100 m m NaCl, 1 m m EGTA, 1 m m Na3 VO4 , 1% NP-40, 1% sodium deoxycholate, 0.01% SDS, and protease inhibitor; Complete, Roche), and biotinylated proteins were precipitated with NeutrAvidin resins (Pierce). .. To collect surface proteins, cells were lysed with lysis buffer (20 m m HEPES, pH 7.5, 100 m m NaCl, 1 m m EGTA, 1 m m Na3 VO4 , 1% NP-40, 1% sodium deoxycholate, 0.01% SDS, and protease inhibitor; Complete, Roche), and biotinylated proteins were precipitated with NeutrAvidin resins (Pierce).

    Avidin-Biotin Assay:

    Article Title: Mechanism for oral tumor cell lysyl oxidase like-2 in cancer development: synergy with PDGF-AB
    Article Snippet: .. Proteins were then subjected to pulldown assays using an avidin-coupled affinity resin (Neutravidin, Pierce Scientific). .. Blots of samples before and after purification were subjected to western blot and visualized with streptavidin-coupled HRP and anti-PDGFRβ antibody to assess for aldehydes and PDGFRβ-aldehydes in response to the CM treatment.

    Labeling:

    Article Title: Polyubiquitination of Prolactin Receptor Stimulates Its Internalization, Postinternalization Sorting, and Degradation via the Lysosomal Pathway ▿Polyubiquitination of Prolactin Receptor Stimulates Its Internalization, Postinternalization Sorting, and Degradation via the Lysosomal Pathway ▿ †
    Article Snippet: .. Lysed cells were transferred to a cold labeled tube, incubated on ice for 20 min, and centrifuged at 15,000 rpm for 15 min. Biotinylated proteins were recovered by incubating with immobilized NeutrAvidin resins (Pierce Inc.) overnight. .. The proteins were washed, boiled in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer, separated by 10% SDS-PAGE, and analyzed by immunoblotting with anti-Flag M2 antibody to detect Flag-tagged PRLr.

    Purification:

    Article Title: Activation-coupled membrane-type 1 matrix metalloproteinase membrane trafficking
    Article Snippet: Paragraph title: Biotinylation and purification of cell-surface proteins ... The labelled proteins were immobilized on NeutrAvidin-conjugated resin (Pierce), and eluted by boiling in Laemmli sample buffer for 15 min before being resolved by SDS/PAGE.

    Article Title: Mechanism for oral tumor cell lysyl oxidase like-2 in cancer development: synergy with PDGF-AB
    Article Snippet: Proteins were then subjected to pulldown assays using an avidin-coupled affinity resin (Neutravidin, Pierce Scientific). .. Blots of samples before and after purification were subjected to western blot and visualized with streptavidin-coupled HRP and anti-PDGFRβ antibody to assess for aldehydes and PDGFRβ-aldehydes in response to the CM treatment.

    Incubation:

    Article Title: Polyubiquitination of Prolactin Receptor Stimulates Its Internalization, Postinternalization Sorting, and Degradation via the Lysosomal Pathway ▿Polyubiquitination of Prolactin Receptor Stimulates Its Internalization, Postinternalization Sorting, and Degradation via the Lysosomal Pathway ▿ †
    Article Snippet: .. Lysed cells were transferred to a cold labeled tube, incubated on ice for 20 min, and centrifuged at 15,000 rpm for 15 min. Biotinylated proteins were recovered by incubating with immobilized NeutrAvidin resins (Pierce Inc.) overnight. .. The proteins were washed, boiled in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer, separated by 10% SDS-PAGE, and analyzed by immunoblotting with anti-Flag M2 antibody to detect Flag-tagged PRLr.

    Cell Culture:

    Article Title: LMTK3 Deficiency Causes Pronounced Locomotor Hyperactivity and Impairs Endocytic Trafficking
    Article Snippet: Paragraph title: Surface biotinylation of cultured neurons ... To collect surface proteins, cells were lysed with lysis buffer (20 m m HEPES, pH 7.5, 100 m m NaCl, 1 m m EGTA, 1 m m Na3 VO4 , 1% NP-40, 1% sodium deoxycholate, 0.01% SDS, and protease inhibitor; Complete, Roche), and biotinylated proteins were precipitated with NeutrAvidin resins (Pierce).

    Biotin Switch Assay:

    Article Title: Thioredoxin-1 Regulates Cellular Heme Insertion by Controlling S-Nitrosation of Glyceraldehyde-3-phosphate Dehydrogenase *
    Article Snippet: .. The biotin switch assay was performed as described , and the pulldowns from NeutrAvidinTM resin (Pierce) were probed by Western blotting. ..

    Western Blot:

    Article Title: Thioredoxin-1 Regulates Cellular Heme Insertion by Controlling S-Nitrosation of Glyceraldehyde-3-phosphate Dehydrogenase *
    Article Snippet: .. The biotin switch assay was performed as described , and the pulldowns from NeutrAvidinTM resin (Pierce) were probed by Western blotting. ..

    Article Title: Mechanism for oral tumor cell lysyl oxidase like-2 in cancer development: synergy with PDGF-AB
    Article Snippet: Proteins were then subjected to pulldown assays using an avidin-coupled affinity resin (Neutravidin, Pierce Scientific). .. Blots of samples before and after purification were subjected to western blot and visualized with streptavidin-coupled HRP and anti-PDGFRβ antibody to assess for aldehydes and PDGFRβ-aldehydes in response to the CM treatment.

    Lysis:

    Article Title: LMTK3 Deficiency Causes Pronounced Locomotor Hyperactivity and Impairs Endocytic Trafficking
    Article Snippet: .. To collect surface proteins, cells were lysed with lysis buffer (20 m m HEPES, pH 7.5, 100 m m NaCl, 1 m m EGTA, 1 m m Na3 VO4 , 1% NP-40, 1% sodium deoxycholate, 0.01% SDS, and protease inhibitor; Complete, Roche), and biotinylated proteins were precipitated with NeutrAvidin resins (Pierce). .. To collect surface proteins, cells were lysed with lysis buffer (20 m m HEPES, pH 7.5, 100 m m NaCl, 1 m m EGTA, 1 m m Na3 VO4 , 1% NP-40, 1% sodium deoxycholate, 0.01% SDS, and protease inhibitor; Complete, Roche), and biotinylated proteins were precipitated with NeutrAvidin resins (Pierce).

    Article Title: Emerging roles of ARHGAP33 in intracellular trafficking of TrkB and pathophysiology of neuropsychiatric disorders
    Article Snippet: .. To collect the surface proteins, cells were lysed with lysis buffer (20 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 0.01% SDS) and biotinylated proteins were precipitated with NeutrAvidin resins (Pierce). ..

    SDS Page:

    Article Title: Polyubiquitination of Prolactin Receptor Stimulates Its Internalization, Postinternalization Sorting, and Degradation via the Lysosomal Pathway ▿Polyubiquitination of Prolactin Receptor Stimulates Its Internalization, Postinternalization Sorting, and Degradation via the Lysosomal Pathway ▿ †
    Article Snippet: Lysed cells were transferred to a cold labeled tube, incubated on ice for 20 min, and centrifuged at 15,000 rpm for 15 min. Biotinylated proteins were recovered by incubating with immobilized NeutrAvidin resins (Pierce Inc.) overnight. .. The proteins were washed, boiled in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer, separated by 10% SDS-PAGE, and analyzed by immunoblotting with anti-Flag M2 antibody to detect Flag-tagged PRLr.

    Article Title: Activation-coupled membrane-type 1 matrix metalloproteinase membrane trafficking
    Article Snippet: .. The labelled proteins were immobilized on NeutrAvidin-conjugated resin (Pierce), and eluted by boiling in Laemmli sample buffer for 15 min before being resolved by SDS/PAGE. .. Transiently transfected COS-7 cells were homogenized in native lysis buffer [50 mM Tris/HCl, pH 7.5, 150 mM NaCl and 1% (v/v) Triton X-100] supplemented with 5 mM EDTA and Complete™ protease inhibitor cocktail (Roche).

    Software:

    Article Title: Polyubiquitination of Prolactin Receptor Stimulates Its Internalization, Postinternalization Sorting, and Degradation via the Lysosomal Pathway ▿Polyubiquitination of Prolactin Receptor Stimulates Its Internalization, Postinternalization Sorting, and Degradation via the Lysosomal Pathway ▿ †
    Article Snippet: Lysed cells were transferred to a cold labeled tube, incubated on ice for 20 min, and centrifuged at 15,000 rpm for 15 min. Biotinylated proteins were recovered by incubating with immobilized NeutrAvidin resins (Pierce Inc.) overnight. .. Densitometry analysis was performed using Image software.

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  • 99
    Thermo Fisher neutravidin
    Course of the in vitro selection. As a measure for the enrichment of biotinylated d -glucagon-binding DNA sequences, the ratio of the fraction of library bound to the target immobilized on streptavidin or <t>neutravidin</t> beads versus the applied biotinylated peptide ( d -glucagon) concentration is plotted against the selection round numbers. A steep increase in binding is visible in rounds 6 to 8 followed by stagnation in rounds 9 to 12. In the following rounds the regular alternation between mutagenic and standard PCR results in alternating ratios.
    Neutravidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher biotin neutravidin bond
    Micro-ring resonator assay for immobilization of Fc-fused Ecad through ProG-TGT on polymer-passivated glass surface. Concentration of reagents: 200 μg/mL <t>neutravidin,</t> 0.05 μM ProG-TGT and 50 μg/mL Ecad. Blue curve is the control run in which PBS buffer was flowed across the surface instead of ProG-TGT solution.
    Biotin Neutravidin Bond, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher hrp conjugated neutravidin
    Targeting strategies and verification of pseudotyped viral coat proteins Schematic representation of viruses conjugated to A) ch128.1 through 2.2 SINDBIS and B) ch128.1Av through biotin-avidin interaction with BAP SINDBIS. C) Western blot analysis of chimeric Sindbis virus envelope proteins. Lentivirus pseudotyped with 2.2 SINDBIS or BAP SINDBIS produced in the presence of pSec BirA and biotin were analyzed using anti-Sindbis ascitic fluid and a secondary anti-mouse <t>IgG-HRP</t> or <t>NeutrAvidin</t> ® -HRP.
    Hrp Conjugated Neutravidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Course of the in vitro selection. As a measure for the enrichment of biotinylated d -glucagon-binding DNA sequences, the ratio of the fraction of library bound to the target immobilized on streptavidin or neutravidin beads versus the applied biotinylated peptide ( d -glucagon) concentration is plotted against the selection round numbers. A steep increase in binding is visible in rounds 6 to 8 followed by stagnation in rounds 9 to 12. In the following rounds the regular alternation between mutagenic and standard PCR results in alternating ratios.

    Journal: The Journal of Biological Chemistry

    Article Title: A Mixed Mirror-image DNA/RNA Aptamer Inhibits Glucagon and Acutely Improves Glucose Tolerance in Models of Type 1 and Type 2 Diabetes *

    doi: 10.1074/jbc.M112.444414

    Figure Lengend Snippet: Course of the in vitro selection. As a measure for the enrichment of biotinylated d -glucagon-binding DNA sequences, the ratio of the fraction of library bound to the target immobilized on streptavidin or neutravidin beads versus the applied biotinylated peptide ( d -glucagon) concentration is plotted against the selection round numbers. A steep increase in binding is visible in rounds 6 to 8 followed by stagnation in rounds 9 to 12. In the following rounds the regular alternation between mutagenic and standard PCR results in alternating ratios.

    Article Snippet: In early rounds, the binding reactions were conducted in solution at 37 °C overnight and in later rounds for 1 to 3 h. Peptide-DNA complexes were immobilized on streptavidin or neutravidin coupled to agarose beads, high capacity agarose plus beads or high capacity streptavidin ultra link plus beads (Thermo Scientific, Rockford, IL), which were added to the reactions at the end of the incubation time.

    Techniques: In Vitro, Selection, Binding Assay, Concentration Assay, Polymerase Chain Reaction

    Site-specifically labeled HA-Srt protein is incorporated into virions. ( A ) Experimental setup. Confluent monolayers of MDCK cells were infected with an MOI = 0.5 during 4.5 hours after which cells were starved and pulse-labeled with [ S 35]Cysteine/Methionine for 20 minutes. After a 2 hour chase, a second pulse-labeling was performed using 100 µM sortase and 250 µM biotin probe to label surface accessible HA-Srt. At indicated timepoints, both cell supernatant as well as cell lysate was analyzed for presence of viral proteins. ( B ) Surface behavior HA on infected MDCK cells analyzed via affinity adsorption to neutravidin-agarose. At indicated timepoint, cells were lysed in 0.5% NP40 buffer and biotin labeled HA-Srt remaining at the cell surface recovered via affinity adsorption on neutravidin-agarose. Proteins were eluted with 2× SDS sample buffer, resolved by 12.5% SDS-PAGE and visualized via autoradiography. ( C ) Accumulation of HA-biotin in supernatant analyzed by affinity adsorption to neutravidin-agarose. Accumulation of biotin-HA-Srt in the supernatant of the cells analyzed in 4B was measured via immunoprecipitation on neutravidin-agarose. Supernatant was lysed via addition of NP40 buffer prior to biding to beads. Proteins were eluted with 2× SDS sample buffer, resolved by 12.5% SDS-PAGE and visualized via autoradiography. ( D ) Quantification of HA loss from the cell surface. Densitometric quantification of radioactivity was performed on autoradiographs from figure 4B and 4C . Total levels of HA-Srt were quantified relative to the levels at the cell surface at t = 0 hrs (top graph). To quantify the kinetics of budding, loss of HA-Srt from the cell surface was quantified as percent reduction relative to the t = 0 timepoint at the cell surface. The rate of accumulation in the cell supernatant was quantified relative to the maximal amount recovered at the t = 10 hrs timepoint. ( E ) Accumulation of whole virus particles analyzed by affinity adsorption to chicken erythrocytes. Accumulation of complete virus particles in the cell supernatant was measured via affinity adsorption on chicken erythrocytes. Supernatant from cells analyzed in 4B was removed at indicated timepoints and mixed with chicken erythrocytes for 30 minutes at 4°C. Cells and bound viral particles were lysed in 2× SDS sample buffer, proteins resolved on 12.5% SDS-PAGE and visualized via autoradiography. ( F ) Kinetcs of virus accumulation as analyzed by adsorption to neutravidin-agarose versus erythrocytes. Densitometric quantification of radioactivity was performed to compare the rate of HA-Srt accumulation in the supernatant compared to whole viral particles (4C versus 4E). Numbers were normalized at t = 1 hrs at which both methods were used.

    Journal: PLoS Pathogens

    Article Title: Chemoenzymatic Site-Specific Labeling of Influenza Glycoproteins as a Tool to Observe Virus Budding in Real Time

    doi: 10.1371/journal.ppat.1002604

    Figure Lengend Snippet: Site-specifically labeled HA-Srt protein is incorporated into virions. ( A ) Experimental setup. Confluent monolayers of MDCK cells were infected with an MOI = 0.5 during 4.5 hours after which cells were starved and pulse-labeled with [ S 35]Cysteine/Methionine for 20 minutes. After a 2 hour chase, a second pulse-labeling was performed using 100 µM sortase and 250 µM biotin probe to label surface accessible HA-Srt. At indicated timepoints, both cell supernatant as well as cell lysate was analyzed for presence of viral proteins. ( B ) Surface behavior HA on infected MDCK cells analyzed via affinity adsorption to neutravidin-agarose. At indicated timepoint, cells were lysed in 0.5% NP40 buffer and biotin labeled HA-Srt remaining at the cell surface recovered via affinity adsorption on neutravidin-agarose. Proteins were eluted with 2× SDS sample buffer, resolved by 12.5% SDS-PAGE and visualized via autoradiography. ( C ) Accumulation of HA-biotin in supernatant analyzed by affinity adsorption to neutravidin-agarose. Accumulation of biotin-HA-Srt in the supernatant of the cells analyzed in 4B was measured via immunoprecipitation on neutravidin-agarose. Supernatant was lysed via addition of NP40 buffer prior to biding to beads. Proteins were eluted with 2× SDS sample buffer, resolved by 12.5% SDS-PAGE and visualized via autoradiography. ( D ) Quantification of HA loss from the cell surface. Densitometric quantification of radioactivity was performed on autoradiographs from figure 4B and 4C . Total levels of HA-Srt were quantified relative to the levels at the cell surface at t = 0 hrs (top graph). To quantify the kinetics of budding, loss of HA-Srt from the cell surface was quantified as percent reduction relative to the t = 0 timepoint at the cell surface. The rate of accumulation in the cell supernatant was quantified relative to the maximal amount recovered at the t = 10 hrs timepoint. ( E ) Accumulation of whole virus particles analyzed by affinity adsorption to chicken erythrocytes. Accumulation of complete virus particles in the cell supernatant was measured via affinity adsorption on chicken erythrocytes. Supernatant from cells analyzed in 4B was removed at indicated timepoints and mixed with chicken erythrocytes for 30 minutes at 4°C. Cells and bound viral particles were lysed in 2× SDS sample buffer, proteins resolved on 12.5% SDS-PAGE and visualized via autoradiography. ( F ) Kinetcs of virus accumulation as analyzed by adsorption to neutravidin-agarose versus erythrocytes. Densitometric quantification of radioactivity was performed to compare the rate of HA-Srt accumulation in the supernatant compared to whole viral particles (4C versus 4E). Numbers were normalized at t = 1 hrs at which both methods were used.

    Article Snippet: Immunoadsportions were performed with 35 µl Neutravidin (Thermo Scientific) agarose for 2 hrs at 4°C after an inital preclear of 4–10 hrs using 120 µl Immobili zed protein A beads (Repligen).

    Techniques: Labeling, Infection, Adsorption, SDS Page, Autoradiography, Immunoprecipitation, Radioactivity

    Micro-ring resonator assay for immobilization of Fc-fused Ecad through ProG-TGT on polymer-passivated glass surface. Concentration of reagents: 200 μg/mL neutravidin, 0.05 μM ProG-TGT and 50 μg/mL Ecad. Blue curve is the control run in which PBS buffer was flowed across the surface instead of ProG-TGT solution.

    Journal: Scientific Reports

    Article Title: Constructing modular and universal single molecule tension sensor using protein G to study mechano-sensitive receptors

    doi: 10.1038/srep21584

    Figure Lengend Snippet: Micro-ring resonator assay for immobilization of Fc-fused Ecad through ProG-TGT on polymer-passivated glass surface. Concentration of reagents: 200 μg/mL neutravidin, 0.05 μM ProG-TGT and 50 μg/mL Ecad. Blue curve is the control run in which PBS buffer was flowed across the surface instead of ProG-TGT solution.

    Article Snippet: To immobilize TGT through a biotin-neutravidin bond on a pegylated glass surface, 200 μg/ml neutravidin (31000, Thermo Fisher Scientific Inc.) in PBS was incubated on such a surface for 20 min.

    Techniques: Concentration Assay

    Targeting strategies and verification of pseudotyped viral coat proteins Schematic representation of viruses conjugated to A) ch128.1 through 2.2 SINDBIS and B) ch128.1Av through biotin-avidin interaction with BAP SINDBIS. C) Western blot analysis of chimeric Sindbis virus envelope proteins. Lentivirus pseudotyped with 2.2 SINDBIS or BAP SINDBIS produced in the presence of pSec BirA and biotin were analyzed using anti-Sindbis ascitic fluid and a secondary anti-mouse IgG-HRP or NeutrAvidin ® -HRP.

    Journal: The journal of gene medicine

    Article Title: Gene delivery in malignant B cells using the combination of lentiviruses conjugated to anti-transferrin receptor antibodies and an immunoglobulin promoter

    doi: 10.1002/jgm.2754

    Figure Lengend Snippet: Targeting strategies and verification of pseudotyped viral coat proteins Schematic representation of viruses conjugated to A) ch128.1 through 2.2 SINDBIS and B) ch128.1Av through biotin-avidin interaction with BAP SINDBIS. C) Western blot analysis of chimeric Sindbis virus envelope proteins. Lentivirus pseudotyped with 2.2 SINDBIS or BAP SINDBIS produced in the presence of pSec BirA and biotin were analyzed using anti-Sindbis ascitic fluid and a secondary anti-mouse IgG-HRP or NeutrAvidin ® -HRP.

    Article Snippet: Biotinylation of envelope proteins was detected using HRP-conjugated NeutrAvidin® (Thermo Fisher Scientific).

    Techniques: Avidin-Biotin Assay, Western Blot, Produced