streptavidin coated magnetic beads  (New England Biolabs)


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    Streptavidin
    Description:
    Streptavidin 1 0 mg
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    n7021s
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    1 0 mg
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    Nucleic Acid Purification Reagents
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    Structured Review

    New England Biolabs streptavidin coated magnetic beads
    Streptavidin
    Streptavidin 1 0 mg
    https://www.bioz.com/result/streptavidin coated magnetic beads/product/New England Biolabs
    Average 97 stars, based on 95 article reviews
    Price from $9.99 to $1999.99
    streptavidin coated magnetic beads - by Bioz Stars, 2021-02
    97/100 stars

    Images

    1) Product Images from "A Novel mRNA Level Subtraction Method for Quick Identification of Target-Orientated Uniquely Expressed Genes Between Peanut Immature Pod and Leaf"

    Article Title: A Novel mRNA Level Subtraction Method for Quick Identification of Target-Orientated Uniquely Expressed Genes Between Peanut Immature Pod and Leaf

    Journal: Biological Procedures Online

    doi: 10.1007/s12575-009-9022-z

    General scheme applied for identifying peanut immature pod-specific genes (tracer mRNA (1)) after a single round subtraction . B biotin, S streptavidin, M magnetic bead.
    Figure Legend Snippet: General scheme applied for identifying peanut immature pod-specific genes (tracer mRNA (1)) after a single round subtraction . B biotin, S streptavidin, M magnetic bead.

    Techniques Used:

    2) Product Images from "Effect of linkers on immobilization of scFvs with biotin-streptavidin interaction"

    Article Title: Effect of linkers on immobilization of scFvs with biotin-streptavidin interaction

    Journal: Biotechnology and applied biochemistry

    doi: 10.1002/bab.1645

    Immobilization of scFv5- linker-BCCP constructs and scFv5-linker-AviTag constructs onto streptavidin-coated plates. Serial dilutions of cell lysates were loaded onto the coated 96-well plates, and the immobilization was detected with an anti-FLAG antibody. Data are normalized to the signal from the highest concentration of scFv5-BCCP with no linker for each biological replicate (n = 6). The error bars represent the standard error of the mean.
    Figure Legend Snippet: Immobilization of scFv5- linker-BCCP constructs and scFv5-linker-AviTag constructs onto streptavidin-coated plates. Serial dilutions of cell lysates were loaded onto the coated 96-well plates, and the immobilization was detected with an anti-FLAG antibody. Data are normalized to the signal from the highest concentration of scFv5-BCCP with no linker for each biological replicate (n = 6). The error bars represent the standard error of the mean.

    Techniques Used: Construct, Concentration Assay

    Immobilization of scFv13R4-linker-BCCP constructs and scFv13R4-linker-AviTag constructs onto streptavidin-coated plates. Serial dilutions of cell lysates were loaded onto the coated 96-well plates, and the immobilization was detected with an anti-FLAG antibody. Data are normalized to the signal from the highest concentration of scFv13R4-BCCP with no linker for each biological replicate (n = 5). The error bars represent the standard error of the mean.
    Figure Legend Snippet: Immobilization of scFv13R4-linker-BCCP constructs and scFv13R4-linker-AviTag constructs onto streptavidin-coated plates. Serial dilutions of cell lysates were loaded onto the coated 96-well plates, and the immobilization was detected with an anti-FLAG antibody. Data are normalized to the signal from the highest concentration of scFv13R4-BCCP with no linker for each biological replicate (n = 5). The error bars represent the standard error of the mean.

    Techniques Used: Construct, Concentration Assay

    3) Product Images from "An efficient and sensitive method for preparing cDNA libraries from scarce biological samples"

    Article Title: An efficient and sensitive method for preparing cDNA libraries from scarce biological samples

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku637

    Overview of 3-day generation of cDNA libraries. ( A ) On the first day, total RNA is ligated to a 3′ adapter and cDNA is generated by reverse transcription by tandem reactions in a single tube, RNA is degraded and cDNAs are isolated by ethanol precipitation. ( B ) On the second day, cDNAs are circularized, size selected by gel fractionation and eluted overnight in the presence of streptavidin beads. ( C ) PCR is done on bead-bound purified cDNAs to generate templates ready for high-throughput sequencing.
    Figure Legend Snippet: Overview of 3-day generation of cDNA libraries. ( A ) On the first day, total RNA is ligated to a 3′ adapter and cDNA is generated by reverse transcription by tandem reactions in a single tube, RNA is degraded and cDNAs are isolated by ethanol precipitation. ( B ) On the second day, cDNAs are circularized, size selected by gel fractionation and eluted overnight in the presence of streptavidin beads. ( C ) PCR is done on bead-bound purified cDNAs to generate templates ready for high-throughput sequencing.

    Techniques Used: Generated, Isolation, Ethanol Precipitation, Fractionation, Polymerase Chain Reaction, Purification, Next-Generation Sequencing

    Detailed LQ cloning method. ( A ) A pre-adenylated (rApp) 3′-terminal dideoxy-C (ddC) blocked adapter (gray) is annealed to a ssDNA reverse transcription (RT) oligo (black) in a 1:1 molar ratio. The annealed adapter is ligated to 3′-hydroxyl-containing RNA (orange) using T4 RNA Ligase 2 (truncated K227Q) without ATP. Each RT oligo contains a 5′ Guanine (G) followed by a 4 or 6 nucleotide randomer (N X ), a 3–6 nucleotide barcode (BAR) and 3 internal deoxyUridine (dU) nucleotides. The adapter::RT oligo hybrid is in excess over RNA, resulting in free adapter::primer material present in the completed reaction. ( B ) Reverse transcription of ligated RNA is carried out in the same tube as the ligation reaction generating ‘+ insert’ and ‘no insert’ cDNA products (red and black line) using dGTP, dTTP, dATP, dCTP as well as biotinylated dATP and dCTP (yellow ‘B’-containing circles). The RNA template is degraded (dashed orange line) by base hydrolysis and cDNA is ethanol precipitated with ammonium acetate to facilitate maximum removal of free adapter and unincorporated nucleotides ( C ). Ethanol precipitated cDNAs are circularized ( D ) and resolved on a 10% denaturing polyacrylamide gel. ‘+ insert’ circularized cDNAs are isolated by excising and eluting them from the gel overnight in the presence of magnetic streptavidin beads ( E ). Bead-bound ‘+ insert’ cDNAs serve as templates in the first round of PCR. Amplification is done using a mix containing uracil-N-deglycosylase (UNG) to remove dU nucleotides, thereby generating a linear template through strand scission, and with primers complimentary to the 3′ adapter (blue) and 5′ end of the RT oligo (tan) ( F ). First round PCR products are resolved on an 8% native polyacrylamide gel, the 60–70 nucleotide products are excised and a portion is used as the template for second round PCR. Second round PCR products are generated using primers complimentary to the 3′ adapter (dark blue) and 5′ end of the RT oligo (brown) that contain the full Illumina or Ion Torrent adapter sequences (dark blue and brown) ( G ).
    Figure Legend Snippet: Detailed LQ cloning method. ( A ) A pre-adenylated (rApp) 3′-terminal dideoxy-C (ddC) blocked adapter (gray) is annealed to a ssDNA reverse transcription (RT) oligo (black) in a 1:1 molar ratio. The annealed adapter is ligated to 3′-hydroxyl-containing RNA (orange) using T4 RNA Ligase 2 (truncated K227Q) without ATP. Each RT oligo contains a 5′ Guanine (G) followed by a 4 or 6 nucleotide randomer (N X ), a 3–6 nucleotide barcode (BAR) and 3 internal deoxyUridine (dU) nucleotides. The adapter::RT oligo hybrid is in excess over RNA, resulting in free adapter::primer material present in the completed reaction. ( B ) Reverse transcription of ligated RNA is carried out in the same tube as the ligation reaction generating ‘+ insert’ and ‘no insert’ cDNA products (red and black line) using dGTP, dTTP, dATP, dCTP as well as biotinylated dATP and dCTP (yellow ‘B’-containing circles). The RNA template is degraded (dashed orange line) by base hydrolysis and cDNA is ethanol precipitated with ammonium acetate to facilitate maximum removal of free adapter and unincorporated nucleotides ( C ). Ethanol precipitated cDNAs are circularized ( D ) and resolved on a 10% denaturing polyacrylamide gel. ‘+ insert’ circularized cDNAs are isolated by excising and eluting them from the gel overnight in the presence of magnetic streptavidin beads ( E ). Bead-bound ‘+ insert’ cDNAs serve as templates in the first round of PCR. Amplification is done using a mix containing uracil-N-deglycosylase (UNG) to remove dU nucleotides, thereby generating a linear template through strand scission, and with primers complimentary to the 3′ adapter (blue) and 5′ end of the RT oligo (tan) ( F ). First round PCR products are resolved on an 8% native polyacrylamide gel, the 60–70 nucleotide products are excised and a portion is used as the template for second round PCR. Second round PCR products are generated using primers complimentary to the 3′ adapter (dark blue) and 5′ end of the RT oligo (brown) that contain the full Illumina or Ion Torrent adapter sequences (dark blue and brown) ( G ).

    Techniques Used: Clone Assay, Ligation, Isolation, Polymerase Chain Reaction, Amplification, Generated

    4) Product Images from ""Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation"

    Article Title: "Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation

    Journal: Analytical chemistry

    doi: 10.1021/ac301278s

    Flow distance traveled as a function of streptavidin concentration in biotin-modified microchannels (58 μm wide) of two different heights, with a 0.5 mm thick PDMS top layer. (A) 13 μm deep channels. (B) 17 μm deep channels.
    Figure Legend Snippet: Flow distance traveled as a function of streptavidin concentration in biotin-modified microchannels (58 μm wide) of two different heights, with a 0.5 mm thick PDMS top layer. (A) 13 μm deep channels. (B) 17 μm deep channels.

    Techniques Used: Flow Cytometry, Concentration Assay, Modification

    Effect of solution viscosity on the flow distance of 100 ng/mL (X) and 1.0 μg/mL (O) streptavidin solutions in biotin-modified 17 μm deep and 58 μm wide channels with a 0.5 mm PDMS top layer. Flow distance is only affected above
    Figure Legend Snippet: Effect of solution viscosity on the flow distance of 100 ng/mL (X) and 1.0 μg/mL (O) streptavidin solutions in biotin-modified 17 μm deep and 58 μm wide channels with a 0.5 mm PDMS top layer. Flow distance is only affected above

    Techniques Used: Flow Cytometry, Modification

    Effect of PDMS layer thickness on the flow distance for 1.0 μg/mL streptavidin in biotin-modified 17 μm deep and 58 μm wide channels. Flow distance decreases asymptotically as PDMS layer thickness is reduced.
    Figure Legend Snippet: Effect of PDMS layer thickness on the flow distance for 1.0 μg/mL streptavidin in biotin-modified 17 μm deep and 58 μm wide channels. Flow distance decreases asymptotically as PDMS layer thickness is reduced.

    Techniques Used: Flow Cytometry, Modification

    Plots of background subtracted, normalized fluorescence signal and peak area in 13 μm deep biotin-modified channels as a function of channel position and flow distance to probe constriction. (A–B) Unlabeled streptavidin (500 μg/mL)
    Figure Legend Snippet: Plots of background subtracted, normalized fluorescence signal and peak area in 13 μm deep biotin-modified channels as a function of channel position and flow distance to probe constriction. (A–B) Unlabeled streptavidin (500 μg/mL)

    Techniques Used: Fluorescence, Modification, Flow Cytometry

    5) Product Images from "Proteins mediating DNA loops effectively block transcription"

    Article Title: Proteins mediating DNA loops effectively block transcription

    Journal: Protein Science : A Publication of the Protein Society

    doi: 10.1002/pro.3156

    LacI bound to an O1 operator pauses transcription. (A) A schematic representation of the DNA template used in magnetic tweezer transcription assays. The template contained a T7A1 promoter close to the upstream end, a stall site at position +22, an O1 operator, the lambda t1 terminator (λt1) and a biotin label at the downstream end. A streptavidin‐labeled paramagnetic bead was coupled to the biotin label to for micromanipulation in the magnetic tweezer. Four examples of transcriptional elongation recorded using the magnetic tweezers are displayed. In ( B ) no LacI was included and transcription shortened the DNA tether progressively without interruption. When LacI was included ( C–E ) , transcription shortened the tether by about 0.2 um before pausing for about 200 s and then resuming. Transcription finally ceased after the tether shortened by either 0.35 um (C and D), a distance corresponding to the location of a terminator sequence, or 0.5 um (B and E), a distance corresponding to the end of the template.
    Figure Legend Snippet: LacI bound to an O1 operator pauses transcription. (A) A schematic representation of the DNA template used in magnetic tweezer transcription assays. The template contained a T7A1 promoter close to the upstream end, a stall site at position +22, an O1 operator, the lambda t1 terminator (λt1) and a biotin label at the downstream end. A streptavidin‐labeled paramagnetic bead was coupled to the biotin label to for micromanipulation in the magnetic tweezer. Four examples of transcriptional elongation recorded using the magnetic tweezers are displayed. In ( B ) no LacI was included and transcription shortened the DNA tether progressively without interruption. When LacI was included ( C–E ) , transcription shortened the tether by about 0.2 um before pausing for about 200 s and then resuming. Transcription finally ceased after the tether shortened by either 0.35 um (C and D), a distance corresponding to the location of a terminator sequence, or 0.5 um (B and E), a distance corresponding to the end of the template.

    Techniques Used: Labeling, Micromanipulation, Sequencing

    Nanographs of RNA polymerases trapped by EDTA quenching during elongation along DNA with and without LacI‐mediated loops. (A) Schematic representation of the DNA templates used in scanning force microscopy assays. All templates contained a T7A1 promoter close to the upstream end, a stall site at position +22, a “far” O1 operator, the lambda t1 terminator (λt1) and a biotin label at the downstream end. The two DNA templates used for SFM measurements of transcription differed in the “near” operator positioned 253 bp downstream from the promoter; one template contained the Os operator while the other contained the O2 operator. The terminator was the very last feature of the sequence and was biotin labeled. Streptavidin was coupled to the biotin label to facilitate identifying the “downstream” end of the molecule in SFM nanographs. (B) The upper row is a selection of molecules along which RNA polymerases (large yellow particle) had not progressed very far from the transcription start site near the end of the DNA without a streptavidin particle (blue). Closed and open conformations of the LacI tetramers are visible for either looped (left) or unlooped (right) columns. The closed conformations are shown as blue particles that are slightly larger than the streptavidin. In the open conformation, two lobes are visible especially on looped DNA. These lobes correspond to individual dimers with DNA binding head groups. The TECs shown in the lower row had progressed further and small coils of RNA emanate from them (see inset schematics for the regions of interest). These TECs have collided with LacI particles. The LacI particles correspond to blue protuberances on the periphery of the larger yellow RNA polymerase particle. The RNA polymerases themselves appear to shift to the side opposite LacI especially for open LacI conformations.
    Figure Legend Snippet: Nanographs of RNA polymerases trapped by EDTA quenching during elongation along DNA with and without LacI‐mediated loops. (A) Schematic representation of the DNA templates used in scanning force microscopy assays. All templates contained a T7A1 promoter close to the upstream end, a stall site at position +22, a “far” O1 operator, the lambda t1 terminator (λt1) and a biotin label at the downstream end. The two DNA templates used for SFM measurements of transcription differed in the “near” operator positioned 253 bp downstream from the promoter; one template contained the Os operator while the other contained the O2 operator. The terminator was the very last feature of the sequence and was biotin labeled. Streptavidin was coupled to the biotin label to facilitate identifying the “downstream” end of the molecule in SFM nanographs. (B) The upper row is a selection of molecules along which RNA polymerases (large yellow particle) had not progressed very far from the transcription start site near the end of the DNA without a streptavidin particle (blue). Closed and open conformations of the LacI tetramers are visible for either looped (left) or unlooped (right) columns. The closed conformations are shown as blue particles that are slightly larger than the streptavidin. In the open conformation, two lobes are visible especially on looped DNA. These lobes correspond to individual dimers with DNA binding head groups. The TECs shown in the lower row had progressed further and small coils of RNA emanate from them (see inset schematics for the regions of interest). These TECs have collided with LacI particles. The LacI particles correspond to blue protuberances on the periphery of the larger yellow RNA polymerase particle. The RNA polymerases themselves appear to shift to the side opposite LacI especially for open LacI conformations.

    Techniques Used: Microscopy, Sequencing, Labeling, Selection, Binding Assay

    Nanographs of RNAP elongation along DNA with and without LacI‐mediated loops. (Top) Schematic representations of transcription elongation complexes (TECs). Columns correspond to different DNA topologies and LacI occupancy. The first column corresponds to transcription elongation without LacI in the reaction buffer. The second and third column correspond to DNA found in a looped topology, with LacI at each of the two operators; the fourth and fifth columns correspond to unlooped DNA with both operators occupied. Transcription elongation progress is categorized in five zones (roman numerals I‐V). Numerals in the schematic correspond to each row of the nanograph array. Each image in the array is representative of its corresponding category (columns) and elongation progress (rows). Image colors indicate height, according to the color scale below. RNAP, LacI and streptavidin particles are indicated by yellow, light blue, and white arrows respectively. (Row I) AFM images of RNAP bound at the T7A1 promoter. (Row II) Images in which TECs have not yet reached the near operator. (Row III) Images in which TECs contact LacI at the near operator. (Row IV) Images in which TECs were found between the two operators. (Row V) Images in which TECs were beyond the far operator. As indicated in the figure, images for RNAP in zone V were not detected for looped O2‐O1 DNA and unlooped Os‐O1 DNA. Note that nascent RNA associated with each TEC is visible, especially in the first column (insets), and increases in size as the RNAP progresses (I to V).
    Figure Legend Snippet: Nanographs of RNAP elongation along DNA with and without LacI‐mediated loops. (Top) Schematic representations of transcription elongation complexes (TECs). Columns correspond to different DNA topologies and LacI occupancy. The first column corresponds to transcription elongation without LacI in the reaction buffer. The second and third column correspond to DNA found in a looped topology, with LacI at each of the two operators; the fourth and fifth columns correspond to unlooped DNA with both operators occupied. Transcription elongation progress is categorized in five zones (roman numerals I‐V). Numerals in the schematic correspond to each row of the nanograph array. Each image in the array is representative of its corresponding category (columns) and elongation progress (rows). Image colors indicate height, according to the color scale below. RNAP, LacI and streptavidin particles are indicated by yellow, light blue, and white arrows respectively. (Row I) AFM images of RNAP bound at the T7A1 promoter. (Row II) Images in which TECs have not yet reached the near operator. (Row III) Images in which TECs contact LacI at the near operator. (Row IV) Images in which TECs were found between the two operators. (Row V) Images in which TECs were beyond the far operator. As indicated in the figure, images for RNAP in zone V were not detected for looped O2‐O1 DNA and unlooped Os‐O1 DNA. Note that nascent RNA associated with each TEC is visible, especially in the first column (insets), and increases in size as the RNAP progresses (I to V).

    Techniques Used:

    6) Product Images from "RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes"

    Article Title: RNA-aptamers-in-droplets (RAPID) high-throughput screening for secretory phenotypes

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00425-7

    RAPID screening for the improvement of streptavidin protein secretion through an evolved secretory tag. The RAPID screening approach was used to identify mutations in the α-mating factor (αMF) secretory leader fused to streptavidin in yeast. a Histograms of droplet fluorescence pre-sort and post-sort (re-encapsulated) demonstrate enrichment through the process. b Isolated and re-transformed clones randomly selected from the pre- and post-sort populations were quantified for streptavidin production. Error bars represent 95% confidence intervals of biological triplicates. Protein secretion from an individual clone was increased nearly threefold over wild-type production using this approach, and there was also twofold increase in secretion between the mean of the sorted and unsorted clones
    Figure Legend Snippet: RAPID screening for the improvement of streptavidin protein secretion through an evolved secretory tag. The RAPID screening approach was used to identify mutations in the α-mating factor (αMF) secretory leader fused to streptavidin in yeast. a Histograms of droplet fluorescence pre-sort and post-sort (re-encapsulated) demonstrate enrichment through the process. b Isolated and re-transformed clones randomly selected from the pre- and post-sort populations were quantified for streptavidin production. Error bars represent 95% confidence intervals of biological triplicates. Protein secretion from an individual clone was increased nearly threefold over wild-type production using this approach, and there was also twofold increase in secretion between the mean of the sorted and unsorted clones

    Techniques Used: Fluorescence, Isolation, Transformation Assay, Clone Assay

    7) Product Images from "La-related protein 1 (LARP1) repression of TOP mRNA translation is mediated through its cap-binding domain and controlled by an adjacent regulatory region"

    Article Title: La-related protein 1 (LARP1) repression of TOP mRNA translation is mediated through its cap-binding domain and controlled by an adjacent regulatory region

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx1237

    The C-terminal half of LARP1 selectively binds TOP sequences and the adjacent cap structure. ( A ) LARP1 497–1019 selectively recognizes oligopyrimidine RNA sequences and the 5′ cap structure. Extracts were prepared from LARP1-null HEK-293T cells expressing either FLAG-tagged LARP1 497-1019 or an N-terminal fragment (1–496) and treated with vehicle (DMSO) or 250 nM Torin 1 for 2 h. Extracts were then incubated with TOP or non-TOP (nTOP) 10 nt RNAs that were either capped or uncapped and containing a 3′ biotin. RNAs were then isolated using streptavidin-coated beads and analyzed by western blotting for the indicated proteins. ( B ) Endogenous LARP1 selectively recognizes capped oligopyrimidine RNA sequences. Extracts were prepared from WT HEK-293T cells treated with DMSO or 250 nM Torin 1 for 2 h, and then incubated with TOP or non-TOP (nTOP) 10 nt RNA probes that were either capped or uncapped and contained a 3′ biotin. RNA probes were isolated as in (A) and analyzed by western blotting for the indicated proteins. ( C ) LARP1 497–1019 fails to interact with PABP. Extracts were prepared from LARP1-null HEK-293T cells expressing either FLAG-tagged LARP1 497-1019 or an N-terminal fragment (1–496) and treated with vehicle (DMSO) or 250 nM Torin 1 for 2 h. FLAG-tagged proteins were then isolated by FLAG-affinity purification in the presence of RNase A, and analyzed by western blotting for the indicated proteins. ( D ) LARP1 mutation that disrupts cap binding prevents TOP mRNA regulation. LARP1-null HEK-293T cells were transfected with the indicated LARP1 cDNAs, along with TOP and non-TOP (nTOP) reporters as in Figure 1D , treated with vehicle (DMSO) or 250 nM Torin 1 for 6 h, and then analyzed for levels of Renilla and firefly luciferase. Data are Renilla/firefly, normalized to vehicle-treated nTOP levels for each LARP1 construct ( n = 3, error bars are SD). ( E ) Expression levels of LARP1 497–1019 WT and Y883A fragments. Cell extracts from cells treated as in (D) were analyzed by western blotting for the indicated proteins.
    Figure Legend Snippet: The C-terminal half of LARP1 selectively binds TOP sequences and the adjacent cap structure. ( A ) LARP1 497–1019 selectively recognizes oligopyrimidine RNA sequences and the 5′ cap structure. Extracts were prepared from LARP1-null HEK-293T cells expressing either FLAG-tagged LARP1 497-1019 or an N-terminal fragment (1–496) and treated with vehicle (DMSO) or 250 nM Torin 1 for 2 h. Extracts were then incubated with TOP or non-TOP (nTOP) 10 nt RNAs that were either capped or uncapped and containing a 3′ biotin. RNAs were then isolated using streptavidin-coated beads and analyzed by western blotting for the indicated proteins. ( B ) Endogenous LARP1 selectively recognizes capped oligopyrimidine RNA sequences. Extracts were prepared from WT HEK-293T cells treated with DMSO or 250 nM Torin 1 for 2 h, and then incubated with TOP or non-TOP (nTOP) 10 nt RNA probes that were either capped or uncapped and contained a 3′ biotin. RNA probes were isolated as in (A) and analyzed by western blotting for the indicated proteins. ( C ) LARP1 497–1019 fails to interact with PABP. Extracts were prepared from LARP1-null HEK-293T cells expressing either FLAG-tagged LARP1 497-1019 or an N-terminal fragment (1–496) and treated with vehicle (DMSO) or 250 nM Torin 1 for 2 h. FLAG-tagged proteins were then isolated by FLAG-affinity purification in the presence of RNase A, and analyzed by western blotting for the indicated proteins. ( D ) LARP1 mutation that disrupts cap binding prevents TOP mRNA regulation. LARP1-null HEK-293T cells were transfected with the indicated LARP1 cDNAs, along with TOP and non-TOP (nTOP) reporters as in Figure 1D , treated with vehicle (DMSO) or 250 nM Torin 1 for 6 h, and then analyzed for levels of Renilla and firefly luciferase. Data are Renilla/firefly, normalized to vehicle-treated nTOP levels for each LARP1 construct ( n = 3, error bars are SD). ( E ) Expression levels of LARP1 497–1019 WT and Y883A fragments. Cell extracts from cells treated as in (D) were analyzed by western blotting for the indicated proteins.

    Techniques Used: Expressing, Incubation, Isolation, Western Blot, Affinity Purification, Mutagenesis, Binding Assay, Transfection, Luciferase, Construct

    8) Product Images from "Simultaneous and stoichiometric purification of hundreds of oligonucleotides"

    Article Title: Simultaneous and stoichiometric purification of hundreds of oligonucleotides

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04870-w

    Stoichiometrically normalizing oligonucleotide purification (SNOP) concept and workflow. a The input reagents for SNOP are chemically synthesized oligonucleotide precursors P 1 through P N that contain imperfect synthesis products with 5′ truncations and/or internal deletions, and with potentially very different concentrations. SNOP produces a pool of oligonucleotide products O 1 through O N that has high fractions of oligos with perfect sequence, and with all products at roughly equal concentration. SNOP uses a single biotinylated capture probe oligonucleotide synthesized with a degenerate “SWSWSW” randomer subsequence. Each instance of the randomer is complementary to one precursor tag sequence. The different instances of the capture probe are all at roughly equal concentration, due to split-pool oligo synthesis. Precursors with perfect tag sequences hybridize to the probe and are captured by streptavidin-coated magnetic beads. Subsequent cleavage at the deoxyuracil (dU) site using the USER enzyme mix ( https://www.neb.com/products/m5505-user-enzyme ) releases the oligo products into solution. Setting the capture probe to be the limiting reagent allows all SNOP products to be all at roughly equal concentrations. b SNOP enriches the fraction of perfect oligos because synthesis errors are correlated; molecules with no truncations or deletions in the tag sequences are also more likely to not have any deletions in the oligo product sequence. Shown in this panel are NGS sequence analysis results of a pool of N = 64 precursor oligonucleotides; error bars show standard deviation across different oligos (see Methods for library preparation details). c SNOP is very sensitive to small sequence changes in the tag; even single-nucleotide variations result in significantly reduced binding yield (see also Supplementary Note). This property allows SNOP products to be both highly pure and stoichiometrically normalized
    Figure Legend Snippet: Stoichiometrically normalizing oligonucleotide purification (SNOP) concept and workflow. a The input reagents for SNOP are chemically synthesized oligonucleotide precursors P 1 through P N that contain imperfect synthesis products with 5′ truncations and/or internal deletions, and with potentially very different concentrations. SNOP produces a pool of oligonucleotide products O 1 through O N that has high fractions of oligos with perfect sequence, and with all products at roughly equal concentration. SNOP uses a single biotinylated capture probe oligonucleotide synthesized with a degenerate “SWSWSW” randomer subsequence. Each instance of the randomer is complementary to one precursor tag sequence. The different instances of the capture probe are all at roughly equal concentration, due to split-pool oligo synthesis. Precursors with perfect tag sequences hybridize to the probe and are captured by streptavidin-coated magnetic beads. Subsequent cleavage at the deoxyuracil (dU) site using the USER enzyme mix ( https://www.neb.com/products/m5505-user-enzyme ) releases the oligo products into solution. Setting the capture probe to be the limiting reagent allows all SNOP products to be all at roughly equal concentrations. b SNOP enriches the fraction of perfect oligos because synthesis errors are correlated; molecules with no truncations or deletions in the tag sequences are also more likely to not have any deletions in the oligo product sequence. Shown in this panel are NGS sequence analysis results of a pool of N = 64 precursor oligonucleotides; error bars show standard deviation across different oligos (see Methods for library preparation details). c SNOP is very sensitive to small sequence changes in the tag; even single-nucleotide variations result in significantly reduced binding yield (see also Supplementary Note). This property allows SNOP products to be both highly pure and stoichiometrically normalized

    Techniques Used: Purification, Synthesized, Sequencing, Concentration Assay, Oligo Synthesis, Magnetic Beads, Next-Generation Sequencing, Standard Deviation, Binding Assay

    9) Product Images from "Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice"

    Article Title: Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice

    Journal: Nature Communications

    doi: 10.1038/ncomms10618

    Binding affinities of the indicated germline VRC01-class antibodies to selected 426c variants. Soluble trimeric 426c gp140 variants were biotinylated and immobilized on a streptavidin biosensor. The association constant of the various germline VRC01-class antibodies was determined by BLI, as described in the Methods section. Undetectable antibody-Env binding is shown on the x axis. Full kinetic parameters are shown in Supplementary Table 1 . See Table 1 for a description of the various mutations on the 426c Env.
    Figure Legend Snippet: Binding affinities of the indicated germline VRC01-class antibodies to selected 426c variants. Soluble trimeric 426c gp140 variants were biotinylated and immobilized on a streptavidin biosensor. The association constant of the various germline VRC01-class antibodies was determined by BLI, as described in the Methods section. Undetectable antibody-Env binding is shown on the x axis. Full kinetic parameters are shown in Supplementary Table 1 . See Table 1 for a description of the various mutations on the 426c Env.

    Techniques Used: Binding Assay

    10) Product Images from "T7 RNA polymerase as a self-replicating label for antigen quantification"

    Article Title: T7 RNA polymerase as a self-replicating label for antigen quantification

    Journal: Nucleic Acids Research

    doi:

    Assay configuration for quantification of antigens using T7RP as a label. The antigen (Ag) is bound simultaneously to an immobilized capture antibody and a biotinylated detection antibody. BT7RP complexed with streptavidin (SA) is then added to the immunocomplex. The bound T7RP is determined by in vitro coupled transcription/translation. Two approaches were explored. (a) T7RP acts on firefly Luc-DNA, located downstream of the T7 promoter, to produce several molecules of active luciferase which is measured by its characteristic bioluminogenic reaction. (b) T7RP acts on T7RP cDNA (T7RP-DNA), positioned downstream of the T7 promoter, to generate several T7RP molecules (self-replication phase) which, in turn, act on Luc-DNA to produce luciferase (detection phase). B, biotin. The T7 promoter is represented by a hatched square.
    Figure Legend Snippet: Assay configuration for quantification of antigens using T7RP as a label. The antigen (Ag) is bound simultaneously to an immobilized capture antibody and a biotinylated detection antibody. BT7RP complexed with streptavidin (SA) is then added to the immunocomplex. The bound T7RP is determined by in vitro coupled transcription/translation. Two approaches were explored. (a) T7RP acts on firefly Luc-DNA, located downstream of the T7 promoter, to produce several molecules of active luciferase which is measured by its characteristic bioluminogenic reaction. (b) T7RP acts on T7RP cDNA (T7RP-DNA), positioned downstream of the T7 promoter, to generate several T7RP molecules (self-replication phase) which, in turn, act on Luc-DNA to produce luciferase (detection phase). B, biotin. The T7 promoter is represented by a hatched square.

    Techniques Used: In Vitro, Luciferase, Activated Clotting Time Assay

    Assessing the sensitivity and analytical range for antigen quantification using T7RP as a label, (squares) without self-replication (absence of T7RP-DNA) and (circles) with self-replication of T7RP. For self-replication, T7RP-DNA was included in the expression reaction mixture and the delayed addition protocol was employed. The immunoassays were carried out as described in Materials and Methods. The luminescence (corrected for the background) is plotted against the concentration of PSA present in the well. The background is defined as the signal obtained in the absence of antigen. Also, shown (triangles) are data for a classical ELISA that uses a streptavidin–alkaline phosphatase conjugate (SA–AP) for detection and p -nitrophenylphosphate as a chromogenic substrate. Following immunocomplex formation (as described in Materials and Methods), SA–AP was added (1000 U), instead of SA–BT7RP, and incubated for 15 min. After washing out the excess of conjugate, the substrate was added for 30 min in the dark, followed by absorbance measurement at 405 nm.
    Figure Legend Snippet: Assessing the sensitivity and analytical range for antigen quantification using T7RP as a label, (squares) without self-replication (absence of T7RP-DNA) and (circles) with self-replication of T7RP. For self-replication, T7RP-DNA was included in the expression reaction mixture and the delayed addition protocol was employed. The immunoassays were carried out as described in Materials and Methods. The luminescence (corrected for the background) is plotted against the concentration of PSA present in the well. The background is defined as the signal obtained in the absence of antigen. Also, shown (triangles) are data for a classical ELISA that uses a streptavidin–alkaline phosphatase conjugate (SA–AP) for detection and p -nitrophenylphosphate as a chromogenic substrate. Following immunocomplex formation (as described in Materials and Methods), SA–AP was added (1000 U), instead of SA–BT7RP, and incubated for 15 min. After washing out the excess of conjugate, the substrate was added for 30 min in the dark, followed by absorbance measurement at 405 nm.

    Techniques Used: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation

    ( A ) Study of the effect of biotinylation on the activity of T7RP. Biotinylation was performed at various NHS-LC-biotin:T7RP molar ratios at pH 7.7 and 9.0, as described in Materials and Methods. T7RP was then determined by in vitro expression using 35 fmol Luc-DNA as template. The percent luminescence is plotted versus the molar ratio NHS-LC-biotin:T7RP at pH 7.7 (solid line) and pH 9.0 (dashed line). The value 100% is defined as the signal obtained from non-biotinylated T7RP. ( B ) Optimization of the streptavidin SA:BT7RP molar ratio for the preparation of the SA–BT7RP complex. The complex was prepared as described in Materials and Methods and 60 fmol were used (without prior purification) for antigen quantification (20 fmol PSA). The solid and dashed lines correspond to the signal and S/B ratio, respectively. The background is defined as the luminescence obtained in the absence of antigen. ( C ) Time dependence of the transcription/translation reaction with T7RP immobilized on the solid phase. The immunoassay was performed as described in Materials and Methods. Following reaction with the SA–BT7RP complex, a 25 µl transcription/translation mixture was added containing 52.5 fmol Luc-DNA as template. Expression was allowed to proceed for various time intervals (up to 180 min). The solid and dashed lines correspond to the signal and S/B ratio, respectively. The background is defined as the luminescence obtained in the absence of antigen. ( D ) Effect of the concentration of SA–BT7RP complex on the luminescence (solid line) and the S/B ratio (dashed line) obtained from the assay of 10 fmol antigen. Luc-DNA was used as template for T7RP. The immunoassay was performed as described in Materials and Methods.
    Figure Legend Snippet: ( A ) Study of the effect of biotinylation on the activity of T7RP. Biotinylation was performed at various NHS-LC-biotin:T7RP molar ratios at pH 7.7 and 9.0, as described in Materials and Methods. T7RP was then determined by in vitro expression using 35 fmol Luc-DNA as template. The percent luminescence is plotted versus the molar ratio NHS-LC-biotin:T7RP at pH 7.7 (solid line) and pH 9.0 (dashed line). The value 100% is defined as the signal obtained from non-biotinylated T7RP. ( B ) Optimization of the streptavidin SA:BT7RP molar ratio for the preparation of the SA–BT7RP complex. The complex was prepared as described in Materials and Methods and 60 fmol were used (without prior purification) for antigen quantification (20 fmol PSA). The solid and dashed lines correspond to the signal and S/B ratio, respectively. The background is defined as the luminescence obtained in the absence of antigen. ( C ) Time dependence of the transcription/translation reaction with T7RP immobilized on the solid phase. The immunoassay was performed as described in Materials and Methods. Following reaction with the SA–BT7RP complex, a 25 µl transcription/translation mixture was added containing 52.5 fmol Luc-DNA as template. Expression was allowed to proceed for various time intervals (up to 180 min). The solid and dashed lines correspond to the signal and S/B ratio, respectively. The background is defined as the luminescence obtained in the absence of antigen. ( D ) Effect of the concentration of SA–BT7RP complex on the luminescence (solid line) and the S/B ratio (dashed line) obtained from the assay of 10 fmol antigen. Luc-DNA was used as template for T7RP. The immunoassay was performed as described in Materials and Methods.

    Techniques Used: Activity Assay, In Vitro, Expressing, Purification, Concentration Assay

    11) Product Images from "PEGylated surfaces for the study of DNA–protein interactions by atomic force microscopy surfaces for the study of DNA–protein interactions by atomic force microscopy †Electronic supplementary information (ESI) available. See DOI: 10.1039/c9nr07104k"

    Article Title: PEGylated surfaces for the study of DNA–protein interactions by atomic force microscopy surfaces for the study of DNA–protein interactions by atomic force microscopy †Electronic supplementary information (ESI) available. See DOI: 10.1039/c9nr07104k

    Journal: Nanoscale

    doi: 10.1039/c9nr07104k

    Streptavidin binding to dual-end biotinylated 672 bp DNA on mica treated with PLL 10 - b -PEG 113 /PLL 1000–2000 . (a) AFM image taken in solution of 672 bp DNA after pre-incubation with ∼50 molar excess of monovalent mono-streptavidin over biotin tag. (b) High-resolution images showing mono-streptavidin bound to both ends of dual-biotin DNA. (c) An AFM image with the colour scale adjusted to highlight immobilized DNA (magenta) and added tetravalent streptavidin (cyan) for streptavidin concentrations of 0 nM, 150 nM and 750 nM (from left to right). Colour scale for (a) and (b) is 5 nm. Scale bars for (a) and (c) are 200 nm and for (b) 50 nm.
    Figure Legend Snippet: Streptavidin binding to dual-end biotinylated 672 bp DNA on mica treated with PLL 10 - b -PEG 113 /PLL 1000–2000 . (a) AFM image taken in solution of 672 bp DNA after pre-incubation with ∼50 molar excess of monovalent mono-streptavidin over biotin tag. (b) High-resolution images showing mono-streptavidin bound to both ends of dual-biotin DNA. (c) An AFM image with the colour scale adjusted to highlight immobilized DNA (magenta) and added tetravalent streptavidin (cyan) for streptavidin concentrations of 0 nM, 150 nM and 750 nM (from left to right). Colour scale for (a) and (b) is 5 nm. Scale bars for (a) and (c) are 200 nm and for (b) 50 nm.

    Techniques Used: Binding Assay, Incubation

    Optimized poly( l -lysine)- b -poly(ethylene glycol) surfaces for exclusive DNA adsorption. AFM images taken in solution showing selective DNA adsorption on PLL 10 - b -PEG 113 /PLL 1000–2000 surfaces. (a) DNA plasmid only. (b) The same area following the addition of 160 nM streptavidin. (c) Percentage background streptavidin coverage at 160 nM for functionalization protocols using different PEG chain lengths, and error bars correspond to the minimum and maximum values as determined from two different areas. (d) A higher resolution image of DNA on the PLL 10 - b -PEG 113 /PLL 1000–2000 surface. Colour scales (see inset top left) for (a) and (b) are 7 nm, (d) with inset colour scale is 9 nm. Scale bars in (a) and (b) are 500 nm and in (d) 200 nm.
    Figure Legend Snippet: Optimized poly( l -lysine)- b -poly(ethylene glycol) surfaces for exclusive DNA adsorption. AFM images taken in solution showing selective DNA adsorption on PLL 10 - b -PEG 113 /PLL 1000–2000 surfaces. (a) DNA plasmid only. (b) The same area following the addition of 160 nM streptavidin. (c) Percentage background streptavidin coverage at 160 nM for functionalization protocols using different PEG chain lengths, and error bars correspond to the minimum and maximum values as determined from two different areas. (d) A higher resolution image of DNA on the PLL 10 - b -PEG 113 /PLL 1000–2000 surface. Colour scales (see inset top left) for (a) and (b) are 7 nm, (d) with inset colour scale is 9 nm. Scale bars in (a) and (b) are 500 nm and in (d) 200 nm.

    Techniques Used: Adsorption, Plasmid Preparation

    Characterization of the adsorption of DNA plasmid and streptavidin on functionalized mica. Streptavidin (160 nM) was added after DNA immobilization, DNA was incubated for 10 minutes prior to imaging and streptavidin was left to equilibrate for 10 minutes prior to imaging on (a) PLL 1000–2000 only surface, (b) PLL 10 - b -PEG 22 and (c) a mixed PLL 10 - b -PEG 22 /PLL 1000–2000 surface. Colour scale (inset in c) 10 nm; scale bar 200 nm. Images taken in solution.
    Figure Legend Snippet: Characterization of the adsorption of DNA plasmid and streptavidin on functionalized mica. Streptavidin (160 nM) was added after DNA immobilization, DNA was incubated for 10 minutes prior to imaging and streptavidin was left to equilibrate for 10 minutes prior to imaging on (a) PLL 1000–2000 only surface, (b) PLL 10 - b -PEG 22 and (c) a mixed PLL 10 - b -PEG 22 /PLL 1000–2000 surface. Colour scale (inset in c) 10 nm; scale bar 200 nm. Images taken in solution.

    Techniques Used: Adsorption, Plasmid Preparation, Incubation, Imaging

    12) Product Images from "Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes *"

    Article Title: Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.730135

    NPH I translocates 5′ to 3′ on single-stranded DNA. A , phosphorimage of a 10% native polyacrylamide gel of a streptavidin displacement assay. Assays were conducted on 36-mer oligonucleotides that were biotinylated at either the 3′
    Figure Legend Snippet: NPH I translocates 5′ to 3′ on single-stranded DNA. A , phosphorimage of a 10% native polyacrylamide gel of a streptavidin displacement assay. Assays were conducted on 36-mer oligonucleotides that were biotinylated at either the 3′

    Techniques Used:

    13) Product Images from "Label-Free Direct Electronic Detection of Biomolecules with Amorphous Silicon Nanostructures"

    Article Title: Label-Free Direct Electronic Detection of Biomolecules with Amorphous Silicon Nanostructures

    Journal:

    doi: 10.1016/j.nano.2006.10.003

    AFM images of flat silicon surface with native oxide before (a) and after (b) surface modification with BAC-BSA and binding to streptavidin and fluorescence imaging confirmation of streptavidin binding to a BAC-BSA-modified native oxide layer (d) but
    Figure Legend Snippet: AFM images of flat silicon surface with native oxide before (a) and after (b) surface modification with BAC-BSA and binding to streptavidin and fluorescence imaging confirmation of streptavidin binding to a BAC-BSA-modified native oxide layer (d) but

    Techniques Used: Modification, BAC Assay, Binding Assay, Fluorescence, Imaging

    measured I–V curves for the sensor before modification, after the attachment of BSA, upon exposure to d-Biotin Streptavidin, and after exposure to Streptavidin. Notice there is a 4.3% average decrease in conductivity after exposure to Streptavidin
    Figure Legend Snippet: measured I–V curves for the sensor before modification, after the attachment of BSA, upon exposure to d-Biotin Streptavidin, and after exposure to Streptavidin. Notice there is a 4.3% average decrease in conductivity after exposure to Streptavidin

    Techniques Used: Modification

    measured transient response of the a-Si sensor to the introduction of streptavidin at t = 360 seconds. The current signal settled to its final value 6 seconds after streptavidin was introduced.
    Figure Legend Snippet: measured transient response of the a-Si sensor to the introduction of streptavidin at t = 360 seconds. The current signal settled to its final value 6 seconds after streptavidin was introduced.

    Techniques Used:

    14) Product Images from ""Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation"

    Article Title: "Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation

    Journal: Analytical chemistry

    doi: 10.1021/ac301278s

    Flow distance traveled as a function of streptavidin concentration in biotin-modified microchannels (58 μm wide) of two different heights, with a 0.5 mm thick PDMS top layer. (A) 13 μm deep channels. (B) 17 μm deep channels.
    Figure Legend Snippet: Flow distance traveled as a function of streptavidin concentration in biotin-modified microchannels (58 μm wide) of two different heights, with a 0.5 mm thick PDMS top layer. (A) 13 μm deep channels. (B) 17 μm deep channels.

    Techniques Used: Flow Cytometry, Concentration Assay, Modification

    Effect of solution viscosity on the flow distance of 100 ng/mL (X) and 1.0 μg/mL (O) streptavidin solutions in biotin-modified 17 μm deep and 58 μm wide channels with a 0.5 mm PDMS top layer. Flow distance is only affected above
    Figure Legend Snippet: Effect of solution viscosity on the flow distance of 100 ng/mL (X) and 1.0 μg/mL (O) streptavidin solutions in biotin-modified 17 μm deep and 58 μm wide channels with a 0.5 mm PDMS top layer. Flow distance is only affected above

    Techniques Used: Flow Cytometry, Modification

    Effect of PDMS layer thickness on the flow distance for 1.0 μg/mL streptavidin in biotin-modified 17 μm deep and 58 μm wide channels. Flow distance decreases asymptotically as PDMS layer thickness is reduced.
    Figure Legend Snippet: Effect of PDMS layer thickness on the flow distance for 1.0 μg/mL streptavidin in biotin-modified 17 μm deep and 58 μm wide channels. Flow distance decreases asymptotically as PDMS layer thickness is reduced.

    Techniques Used: Flow Cytometry, Modification

    Plots of background subtracted, normalized fluorescence signal and peak area in 13 μm deep biotin-modified channels as a function of channel position and flow distance to probe constriction. (A–B) Unlabeled streptavidin (500 μg/mL)
    Figure Legend Snippet: Plots of background subtracted, normalized fluorescence signal and peak area in 13 μm deep biotin-modified channels as a function of channel position and flow distance to probe constriction. (A–B) Unlabeled streptavidin (500 μg/mL)

    Techniques Used: Fluorescence, Modification, Flow Cytometry

    15) Product Images from "Mismatch repair and DNA polymerase δ proofreading prevent catastrophic accumulation of leading strand errors in cells expressing a cancer-associated DNA polymerase ϵ variant"

    Article Title: Mismatch repair and DNA polymerase δ proofreading prevent catastrophic accumulation of leading strand errors in cells expressing a cancer-associated DNA polymerase ϵ variant

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa633

    A bias in the formation of reciprocal mispairs at the ura3-29 mutation site. ( A ) Oligonucleotide substrates for primer extension assays. The DNA sequence of the substrates corresponds to the sequence context of the ura3-29 mutation. Sequences of the non-transcribed and transcribed strands serve as templates in the top and bottom substrates, respectively. The mutation site is indicated. For complete primer and template sequences, see Materials and Methods. Streptavidin bumpers are shown as grey circles. ( B ) Primer extension by Polϵ-exo − and Polϵ-P301R on substrates described in (A). Reactions were carried out for 5 min using a 4:1 ratio of substrate to polymerase, and the products were separated by denaturing polyacrylamide gel electrophoresis. The dNTPs present in each reaction are indicated below the gel image. ( C ) The efficiency of nucleotide misincorporation by Polϵ-exo − and Polϵ-P301R at the ura3-29 mutation site. Percent misincorporation was calculated by dividing the fraction of primer extended with an incorrect nucleotide by the fraction of primer extended with the correct nucleotide. Data are averages of three experiments. Error bars represent standard deviation.
    Figure Legend Snippet: A bias in the formation of reciprocal mispairs at the ura3-29 mutation site. ( A ) Oligonucleotide substrates for primer extension assays. The DNA sequence of the substrates corresponds to the sequence context of the ura3-29 mutation. Sequences of the non-transcribed and transcribed strands serve as templates in the top and bottom substrates, respectively. The mutation site is indicated. For complete primer and template sequences, see Materials and Methods. Streptavidin bumpers are shown as grey circles. ( B ) Primer extension by Polϵ-exo − and Polϵ-P301R on substrates described in (A). Reactions were carried out for 5 min using a 4:1 ratio of substrate to polymerase, and the products were separated by denaturing polyacrylamide gel electrophoresis. The dNTPs present in each reaction are indicated below the gel image. ( C ) The efficiency of nucleotide misincorporation by Polϵ-exo − and Polϵ-P301R at the ura3-29 mutation site. Percent misincorporation was calculated by dividing the fraction of primer extended with an incorrect nucleotide by the fraction of primer extended with the correct nucleotide. Data are averages of three experiments. Error bars represent standard deviation.

    Techniques Used: Mutagenesis, Sequencing, Polyacrylamide Gel Electrophoresis, Standard Deviation

    16) Product Images from "Poly(A)-tail profiling reveals an embryonic switch in translational control"

    Article Title: Poly(A)-tail profiling reveals an embryonic switch in translational control

    Journal: Nature

    doi: 10.1038/nature13007

    Global measurement of poly(A)-tail lengths a , Outline of PAL-seq. For each cluster, the fluorescence intensity reflects the tail length of the cDNA that seeded the cluster. Although the probability of incorporating a biotin-conjugated dU opposite each tail nucleotide is uniform, stochastic incorporation results in a variable number of biotins for each molecule within a cluster. b , Median streptavidin fluorescence intensities for two sets of mRNA-like molecules with indicated poly(A)-tail lengths, which were added to 3T3 (circle), HEK293T (triangle), and HeLa (square) samples for tail-length calibration.
    Figure Legend Snippet: Global measurement of poly(A)-tail lengths a , Outline of PAL-seq. For each cluster, the fluorescence intensity reflects the tail length of the cDNA that seeded the cluster. Although the probability of incorporating a biotin-conjugated dU opposite each tail nucleotide is uniform, stochastic incorporation results in a variable number of biotins for each molecule within a cluster. b , Median streptavidin fluorescence intensities for two sets of mRNA-like molecules with indicated poly(A)-tail lengths, which were added to 3T3 (circle), HEK293T (triangle), and HeLa (square) samples for tail-length calibration.

    Techniques Used: Fluorescence

    17) Product Images from "A DNA nanoscope via auto-cycling proximity recording"

    Article Title: A DNA nanoscope via auto-cycling proximity recording

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00542-3

    Proof-of-principle experiments. a Generation of Full-records requires the colocalization of probes, here by biotinylated hairpin loops bound to streptavidin, whereas isolated probes can always generate Half-records. Cropped denaturing PAGE gel depicting 10 μl reactions (40 min at 37 °C) with biotin–streptavidin association and 4:1 overall probe/streptavidin stoichiometry ( inset ), 8 and 22 nt barcodes (19 and 33 nt stem lengths copied), 10:1 initial primer/probe, and 40 nM total probe concentration. A single primer sequence was used and no secondary amplification was performed. b Auto-cycling is demonstrated by quantification of Cy5-labeled probes on cropped denaturing PAGE gels. Rapidly cycling probes with Iso-dC/dG stoppers and phosphorothioate primers were used, with probes at 0.1 nM and primers at 1000-fold excess to probes in each time series. Quantification of Full-records yields the plot. Half-records are difficult to detect because of low probe concentration. See probe details and sequences for a , b in Supplementary Fig. 2 and full gels in Supplementary Fig. 3 . c An example of a single probe (with Barcode i ) making multiple partnerships (with Barcodes \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${j^*}$$\end{document} j * ), read with Illumina MiSeq next-generation sequencing. Here, probes encoded a universal primer sequence and unique barcodes (in place of spacer s of Fig. 2a ), and were held in tetramers by streptavidin. Primer sequences cropped for clarity. See Supplementary Fig. 4 for the unique-barcode APR cycle, probe details, and sequencing methods
    Figure Legend Snippet: Proof-of-principle experiments. a Generation of Full-records requires the colocalization of probes, here by biotinylated hairpin loops bound to streptavidin, whereas isolated probes can always generate Half-records. Cropped denaturing PAGE gel depicting 10 μl reactions (40 min at 37 °C) with biotin–streptavidin association and 4:1 overall probe/streptavidin stoichiometry ( inset ), 8 and 22 nt barcodes (19 and 33 nt stem lengths copied), 10:1 initial primer/probe, and 40 nM total probe concentration. A single primer sequence was used and no secondary amplification was performed. b Auto-cycling is demonstrated by quantification of Cy5-labeled probes on cropped denaturing PAGE gels. Rapidly cycling probes with Iso-dC/dG stoppers and phosphorothioate primers were used, with probes at 0.1 nM and primers at 1000-fold excess to probes in each time series. Quantification of Full-records yields the plot. Half-records are difficult to detect because of low probe concentration. See probe details and sequences for a , b in Supplementary Fig. 2 and full gels in Supplementary Fig. 3 . c An example of a single probe (with Barcode i ) making multiple partnerships (with Barcodes \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${j^*}$$\end{document} j * ), read with Illumina MiSeq next-generation sequencing. Here, probes encoded a universal primer sequence and unique barcodes (in place of spacer s of Fig. 2a ), and were held in tetramers by streptavidin. Primer sequences cropped for clarity. See Supplementary Fig. 4 for the unique-barcode APR cycle, probe details, and sequencing methods

    Techniques Used: Isolation, Polyacrylamide Gel Electrophoresis, Concentration Assay, Sequencing, Amplification, Labeling, Next-Generation Sequencing

    18) Product Images from "An RNA polymerase ribozyme that synthesizes its own ancestor"

    Article Title: An RNA polymerase ribozyme that synthesizes its own ancestor

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

    doi: 10.1073/pnas.1914282117

    In vitro evolution of the 38-6 RNA polymerase ribozyme. ( A ) Scheme for selective amplification of polymerase ribozymes that synthesize a functional hammerhead ribozyme. (1) Attachment to the polymerase of an RNA primer (magenta), biotin (green), and the RNA substrate (orange) to be cleaved by the hammerhead. (2) Hybridization of the primer to an RNA template (brown) that encodes the hammerhead. (3) Extension of the primer by polymerization of NTPs (cyan), followed by biotin capture on streptavidin magnetic beads (gray). (4) Cleavage of the attached RNA substrate by the hammerhead, releasing the polymerase from the beads. (5) Recovery of functional polymerases. (6) Reverse transcription and PCR amplification. (7) Transcription to generate progeny polymerases. ( B ) Sequence and secondary structure of the hammerhead ribozyme (cyan), together with the primer used to initiate its synthesis and the RNA substrate. The arrow indicates the site of cleavage. ( C ). Stem elements P3–P7 within the core domain are labeled.
    Figure Legend Snippet: In vitro evolution of the 38-6 RNA polymerase ribozyme. ( A ) Scheme for selective amplification of polymerase ribozymes that synthesize a functional hammerhead ribozyme. (1) Attachment to the polymerase of an RNA primer (magenta), biotin (green), and the RNA substrate (orange) to be cleaved by the hammerhead. (2) Hybridization of the primer to an RNA template (brown) that encodes the hammerhead. (3) Extension of the primer by polymerization of NTPs (cyan), followed by biotin capture on streptavidin magnetic beads (gray). (4) Cleavage of the attached RNA substrate by the hammerhead, releasing the polymerase from the beads. (5) Recovery of functional polymerases. (6) Reverse transcription and PCR amplification. (7) Transcription to generate progeny polymerases. ( B ) Sequence and secondary structure of the hammerhead ribozyme (cyan), together with the primer used to initiate its synthesis and the RNA substrate. The arrow indicates the site of cleavage. ( C ). Stem elements P3–P7 within the core domain are labeled.

    Techniques Used: In Vitro, Amplification, Functional Assay, Hybridization, Magnetic Beads, Polymerase Chain Reaction, Sequencing, Labeling

    19) Product Images from "mTORC1 Balances Cellular Amino Acid Supply with Demand for Protein Synthesis through Post-transcriptional Control of ATF4"

    Article Title: mTORC1 Balances Cellular Amino Acid Supply with Demand for Protein Synthesis through Post-transcriptional Control of ATF4

    Journal: Cell reports

    doi: 10.1016/j.celrep.2017.04.042

    mTOR Controls ATF4 Translation and mRNA Stability (A) mTOR reduces ATF4 mRNA levels. RNA was isolated from cells treated with vehicle (DMSO) or 250 nM Torin 1 for the indicated times and analyzed by qPCR. RNA levels were normalized to GAPDH (n = 3, error bars are SD). (B) mTOR activity has little effect on ATF4 transcription. HEK293T cells were treated with vehicle or 250 nM Torin 1 for 4 hr and then pulsed for 15 and 30 min with 100 μM 4sU. RNA was reacted with MTS-biotin, isolated by streptavidin-affinity purification, and analyzed by qPCR. Synthesis rates were determined by comparing 4sU labeling at 15 and 30 min and compared to changes in steady-state mRNA levels (n = 3, error bars are SD). (C) mTOR inhibition decreases the half-life of ATF4 mRNA. ATF4 −/− HEK293T cells simultaneously expressing doxycycline-repressible constructs encoding ATF4 and GFP were pre-treated with vehicle or 250 nM Torin 1 for 30 min, and then 1 μg/mL doxycycline. mRNA was collected at 0 and 6 hr post-doxycycline addition and analyzed by qPCR. mRNA levels were normalized to GAPDH (n = 3, error bars are SD, but are too small to be visible). (D) ATF4 protein stability is unaffected by mTOR inhibition. Extracts were prepared from HEK293T cells pre-treated with 100 μg/mL cycloheximide for 5 min and then with vehicle (DMSO) or 250 nM Torin 1 for the indicated times, and they were analyzed for the indicated proteins by immunoblotting (left panel) and quantified by normalizing levels of ATF4 to EIF3B (right panel) (n = 3, error bars are SD). (E) mTOR inhibition preferentially decreases translation of the ATF4-coding ORF. Top panel: ribosome profiling data from HEK293T cells treated for 24 hr with vehicle (DMSO) or 250 nM Torin 1 are shown. Bar heights are reads per million (RPM) for each position in the spliced ATF4 transcript, and they are the combined values of two replicate libraries. Bottom panel: organization of ORFs in the ATF4 mRNA is shown. (F) mTOR-regulated change in the translation efficiency of ATF4 ORFs. Translation efficiencies of ATF4 uORF3 and main ORF (mORF) were calculated by normalizing ribosome-protected fragment (RPF) reads from (E) from non-overlapping segments of uORF3 or mORF to RNA levels in DMSO- and Torin 1-treated conditions (n = 2, error bars are SD, significance calculated by t test). (G) Top panel: reporter design. 5′ UTRs are from wild-type human ATF4 (WT), ATF4 with start codon of uORF3 mutated to TAC (DuORF3), or ACTB. Bottom panel: cells were treated with 10 μM TMP to stabilize YFP concurrently with vehicle (DMSO) or 250 nM Torin 1, and they were monitored for fluorescence at the indicated times (n = 9, error bars are SEM). (H) uORF3 is required for mTOR control of full-length ATF4. ATF4 −/− HEK293T cells stably expressing dox-inducible WT or DuORF3 ATF4 were treated with 1.0 μg/mL (WT) or 0.5 μg/mL (ΔuORF3) doxycycline for 40 hr, and then with vehicle (DMSO) or 250 nM Torin 1 for 1 hr. Cell extracts were prepared and analyzed by immunoblotting for the indicated proteins. (I) Gcn2 is required for mTOR control of eIF2α phosphorylation, but not ATF4 translation. Extracts were prepared from Gcn2 +/+ or Gcn2 −/− MEFs treated with vehicle (DMSO) or 250 nM Torin 1 for 4 hr, and they were analyzed by immunoblotting for the indicated proteins.
    Figure Legend Snippet: mTOR Controls ATF4 Translation and mRNA Stability (A) mTOR reduces ATF4 mRNA levels. RNA was isolated from cells treated with vehicle (DMSO) or 250 nM Torin 1 for the indicated times and analyzed by qPCR. RNA levels were normalized to GAPDH (n = 3, error bars are SD). (B) mTOR activity has little effect on ATF4 transcription. HEK293T cells were treated with vehicle or 250 nM Torin 1 for 4 hr and then pulsed for 15 and 30 min with 100 μM 4sU. RNA was reacted with MTS-biotin, isolated by streptavidin-affinity purification, and analyzed by qPCR. Synthesis rates were determined by comparing 4sU labeling at 15 and 30 min and compared to changes in steady-state mRNA levels (n = 3, error bars are SD). (C) mTOR inhibition decreases the half-life of ATF4 mRNA. ATF4 −/− HEK293T cells simultaneously expressing doxycycline-repressible constructs encoding ATF4 and GFP were pre-treated with vehicle or 250 nM Torin 1 for 30 min, and then 1 μg/mL doxycycline. mRNA was collected at 0 and 6 hr post-doxycycline addition and analyzed by qPCR. mRNA levels were normalized to GAPDH (n = 3, error bars are SD, but are too small to be visible). (D) ATF4 protein stability is unaffected by mTOR inhibition. Extracts were prepared from HEK293T cells pre-treated with 100 μg/mL cycloheximide for 5 min and then with vehicle (DMSO) or 250 nM Torin 1 for the indicated times, and they were analyzed for the indicated proteins by immunoblotting (left panel) and quantified by normalizing levels of ATF4 to EIF3B (right panel) (n = 3, error bars are SD). (E) mTOR inhibition preferentially decreases translation of the ATF4-coding ORF. Top panel: ribosome profiling data from HEK293T cells treated for 24 hr with vehicle (DMSO) or 250 nM Torin 1 are shown. Bar heights are reads per million (RPM) for each position in the spliced ATF4 transcript, and they are the combined values of two replicate libraries. Bottom panel: organization of ORFs in the ATF4 mRNA is shown. (F) mTOR-regulated change in the translation efficiency of ATF4 ORFs. Translation efficiencies of ATF4 uORF3 and main ORF (mORF) were calculated by normalizing ribosome-protected fragment (RPF) reads from (E) from non-overlapping segments of uORF3 or mORF to RNA levels in DMSO- and Torin 1-treated conditions (n = 2, error bars are SD, significance calculated by t test). (G) Top panel: reporter design. 5′ UTRs are from wild-type human ATF4 (WT), ATF4 with start codon of uORF3 mutated to TAC (DuORF3), or ACTB. Bottom panel: cells were treated with 10 μM TMP to stabilize YFP concurrently with vehicle (DMSO) or 250 nM Torin 1, and they were monitored for fluorescence at the indicated times (n = 9, error bars are SEM). (H) uORF3 is required for mTOR control of full-length ATF4. ATF4 −/− HEK293T cells stably expressing dox-inducible WT or DuORF3 ATF4 were treated with 1.0 μg/mL (WT) or 0.5 μg/mL (ΔuORF3) doxycycline for 40 hr, and then with vehicle (DMSO) or 250 nM Torin 1 for 1 hr. Cell extracts were prepared and analyzed by immunoblotting for the indicated proteins. (I) Gcn2 is required for mTOR control of eIF2α phosphorylation, but not ATF4 translation. Extracts were prepared from Gcn2 +/+ or Gcn2 −/− MEFs treated with vehicle (DMSO) or 250 nM Torin 1 for 4 hr, and they were analyzed by immunoblotting for the indicated proteins.

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Activity Assay, Affinity Purification, Labeling, Inhibition, Expressing, Construct, Fluorescence, Stable Transfection

    20) Product Images from "The MinDE system is a generic spatial cue for membrane protein distribution in vitro"

    Article Title: The MinDE system is a generic spatial cue for membrane protein distribution in vitro

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06310-1

    MinDE spatiotemporally regulate membrane-anchored DNA. a MinDE self-organization can regulate short membrane-anchored DNA fragments. Representative images and kymograph of a time-series of MinDE self-organization in the presence of 30 bp P1 dsDNA bound to the membrane by a cholesterol anchor (1 µM MinD (30% EGFP-MinD), 1 µM MinE, 10 nM TEG-cholesterol-dsP1). b Representative images and kymograph of a time-series of MinDE self-organization spatiotemporally regulating 300 bp long dsDNA bound to lipid-anchored streptavidin (1 µM MinD (30% EGFP-MinD), 1 µM MinE, 300 bp lambda DNA, streptavidin). c Representative images and kymograph of a time-series of MinDE self-organization spatiotemporally regulating 2000 bp long dsDNA bound to lipid-anchored streptavidin. All experiments were performed independently two ( c ) or three ( a , b ) times under similar conditions. Scale bars: 50 µm
    Figure Legend Snippet: MinDE spatiotemporally regulate membrane-anchored DNA. a MinDE self-organization can regulate short membrane-anchored DNA fragments. Representative images and kymograph of a time-series of MinDE self-organization in the presence of 30 bp P1 dsDNA bound to the membrane by a cholesterol anchor (1 µM MinD (30% EGFP-MinD), 1 µM MinE, 10 nM TEG-cholesterol-dsP1). b Representative images and kymograph of a time-series of MinDE self-organization spatiotemporally regulating 300 bp long dsDNA bound to lipid-anchored streptavidin (1 µM MinD (30% EGFP-MinD), 1 µM MinE, 300 bp lambda DNA, streptavidin). c Representative images and kymograph of a time-series of MinDE self-organization spatiotemporally regulating 2000 bp long dsDNA bound to lipid-anchored streptavidin. All experiments were performed independently two ( c ) or three ( a , b ) times under similar conditions. Scale bars: 50 µm

    Techniques Used: Lambda DNA Preparation

    MinDE-driven dynamics of model membrane proteins in vitro suggest that MinDE form a propagating diffusion barrier. a Representative images and kymographs of colliding MinDE waves in the presence of mCh-MTS(BsD) and lipid-anchored streptavidin bound to biotinylated lipids (1 µM MinD (30% EGFP-MinD), 1 µM MinE, 1 µM mCh-MTS(BsD) or streptavidin-Alexa647). Scale bars: 50 µm. b Schematic of the underlying protein behavior resulting in spatiotemporal regulation of model peripheral and membrane-anchored proteins. While mCh-MTS and MinDE can also attach and detach to and from the membrane, streptavidin can only diffuse laterally on the membrane. Schematic density profiles and protein localization on the membrane (magenta: mCh-MTS, green: MinD, orange: MinE, cyan: lipid-anchored streptavidin). The MinDE wave propagates directionally, even if individual proteins show a random movement on the membrane. Both model peripheral and membrane-anchored proteins show a wave propagation in the direction of the MinDE wave. mCh-MTS while more abundant in the MinDE minima covers the membrane homogenously. In contrast the resulting secondary wave of streptavidin shows an inhomogeneous profile and results in a net transport of the membrane-anchored protein
    Figure Legend Snippet: MinDE-driven dynamics of model membrane proteins in vitro suggest that MinDE form a propagating diffusion barrier. a Representative images and kymographs of colliding MinDE waves in the presence of mCh-MTS(BsD) and lipid-anchored streptavidin bound to biotinylated lipids (1 µM MinD (30% EGFP-MinD), 1 µM MinE, 1 µM mCh-MTS(BsD) or streptavidin-Alexa647). Scale bars: 50 µm. b Schematic of the underlying protein behavior resulting in spatiotemporal regulation of model peripheral and membrane-anchored proteins. While mCh-MTS and MinDE can also attach and detach to and from the membrane, streptavidin can only diffuse laterally on the membrane. Schematic density profiles and protein localization on the membrane (magenta: mCh-MTS, green: MinD, orange: MinE, cyan: lipid-anchored streptavidin). The MinDE wave propagates directionally, even if individual proteins show a random movement on the membrane. Both model peripheral and membrane-anchored proteins show a wave propagation in the direction of the MinDE wave. mCh-MTS while more abundant in the MinDE minima covers the membrane homogenously. In contrast the resulting secondary wave of streptavidin shows an inhomogeneous profile and results in a net transport of the membrane-anchored protein

    Techniques Used: In Vitro, Diffusion-based Assay

    MinDE induce oscillatory and time-averaged concentration gradients of model membrane proteins in microcompartments. a Experimental setup: PDMS-microcompartments are lined with an SLB and covered by air to confine the proteins. b Representative time-lapse images and kymographs of MinDE oscillations and streptavidin counter-oscillations in the compartments (1 µM MinD, 2 µM MinE, streptavidin-Alexa647). Brightness of the streptavidin channel was corrected for bleaching using histogram matching in Fiji. Scale bars: 10 µm. c Time-averaged fluorescence intensity profiles of MinDE (green) and streptavidin (cyan) oscillation in b showing clear concentration gradients for both MinD and streptavidin. d Time-averaged fluorescence intensity profiles (gray lines) for EGFP-MinD and streptavidin aligned and projected to a unit box (see Supplementary Fig. 14 for details). Bold, colored lines represent the mean profiles, generated from three independent experiments with N = 35 microcompartments. e Representative time-lapse images and kymographs of MinDE oscillations and mCh-MTS(BsD) counter-oscillations in PDMS microcompartments (1 µM MinD (30% EGFP-MinD), 2 µM MinE, 0.5 µM mCh-MTS(BsD)). Scale bars: 10 µm. f Time-averaged fluorescence intensity profiles of MinDE (green) and mCh-MTS(BsD) (magenta) oscillations in e showing a clear protein gradient for MinD and homogenous protein distribution of mCh-MTS(BsD). g Time-averaged fluorescence intensity profiles (gray lines) for EGFP-MinD and mCh-MTS(BsD) aligned and projected to a unit box. Bold, colored lines represent the mean profiles, generated from three independent experiments with in total N = 45 microcompartments. h Schematic explaining how the MinDE system positions lipid-anchored streptavidin and mCh-MTS constructs in rod-shaped microcompartments. MinDE oscillations drive counter-oscillations of lipid-anchored streptavidin and mCh-MTS constructs, thereby establishing a time-averaged concentration gradient of lipid-anchored streptavidin with maximal concentration in the geometric center, but no concentration gradient of mCh-MTS
    Figure Legend Snippet: MinDE induce oscillatory and time-averaged concentration gradients of model membrane proteins in microcompartments. a Experimental setup: PDMS-microcompartments are lined with an SLB and covered by air to confine the proteins. b Representative time-lapse images and kymographs of MinDE oscillations and streptavidin counter-oscillations in the compartments (1 µM MinD, 2 µM MinE, streptavidin-Alexa647). Brightness of the streptavidin channel was corrected for bleaching using histogram matching in Fiji. Scale bars: 10 µm. c Time-averaged fluorescence intensity profiles of MinDE (green) and streptavidin (cyan) oscillation in b showing clear concentration gradients for both MinD and streptavidin. d Time-averaged fluorescence intensity profiles (gray lines) for EGFP-MinD and streptavidin aligned and projected to a unit box (see Supplementary Fig. 14 for details). Bold, colored lines represent the mean profiles, generated from three independent experiments with N = 35 microcompartments. e Representative time-lapse images and kymographs of MinDE oscillations and mCh-MTS(BsD) counter-oscillations in PDMS microcompartments (1 µM MinD (30% EGFP-MinD), 2 µM MinE, 0.5 µM mCh-MTS(BsD)). Scale bars: 10 µm. f Time-averaged fluorescence intensity profiles of MinDE (green) and mCh-MTS(BsD) (magenta) oscillations in e showing a clear protein gradient for MinD and homogenous protein distribution of mCh-MTS(BsD). g Time-averaged fluorescence intensity profiles (gray lines) for EGFP-MinD and mCh-MTS(BsD) aligned and projected to a unit box. Bold, colored lines represent the mean profiles, generated from three independent experiments with in total N = 45 microcompartments. h Schematic explaining how the MinDE system positions lipid-anchored streptavidin and mCh-MTS constructs in rod-shaped microcompartments. MinDE oscillations drive counter-oscillations of lipid-anchored streptavidin and mCh-MTS constructs, thereby establishing a time-averaged concentration gradient of lipid-anchored streptavidin with maximal concentration in the geometric center, but no concentration gradient of mCh-MTS

    Techniques Used: Concentration Assay, Fluorescence, Generated, Construct

    MinDE spatiotemporally position a lipid-anchored protein resulting in large-scale concentration gradients. a MinDE self-organization spatiotemporally regulates lipid-anchored streptavidin. Representative time series of MinDE self-organization on a SLB with Biotinyl-CAP-PE-bound streptavidin (1 µM MinD, 1 µM MinE, streptavidin-Alexa647). ATP is added at t = 0 s to start self-organization. Scale bars: 50 µm. b Schematic of the experimental setup. Tetrameric streptavidin is anchored to the SLB by binding two to three Biotinyl-CAP-PE lipids and MinDE and ATP are added. c Kymograph of the line selections shown in a . Scale bars: 50 µm and 10 min. d MinDE self-organization leads to large-scale concentration gradients of streptavidin. Representative images of streptavidin distribution in MinDE spirals after > 1 h of MinDE self-organization on SLBs. Fluorescence intensity line plots of EGFP-MinD and streptavidin distribution of selections shown in the respective images. Scale bars: 50 µm. e Large-scale streptavidin gradient formation by MinDE is reversible. Representative images and kymograph (1) of a running MinDE assay in the presence of anchored streptavidin. Addition of sodium orthovanadate (Na 3 VO 4 ) leads to MinDE detachment which in turn leads to homogenization of streptavidin fluorescence on the membrane. Fluorescence intensity of streptavidin (cyan) and EGFP-MinD (green) is plotted over the duration of the time-lapse in the center (2) and at the rim of the MinDE spiral (3). Scale bars: 50 µm and 300 s. All experiments were performed independently three or more times under identical conditions
    Figure Legend Snippet: MinDE spatiotemporally position a lipid-anchored protein resulting in large-scale concentration gradients. a MinDE self-organization spatiotemporally regulates lipid-anchored streptavidin. Representative time series of MinDE self-organization on a SLB with Biotinyl-CAP-PE-bound streptavidin (1 µM MinD, 1 µM MinE, streptavidin-Alexa647). ATP is added at t = 0 s to start self-organization. Scale bars: 50 µm. b Schematic of the experimental setup. Tetrameric streptavidin is anchored to the SLB by binding two to three Biotinyl-CAP-PE lipids and MinDE and ATP are added. c Kymograph of the line selections shown in a . Scale bars: 50 µm and 10 min. d MinDE self-organization leads to large-scale concentration gradients of streptavidin. Representative images of streptavidin distribution in MinDE spirals after > 1 h of MinDE self-organization on SLBs. Fluorescence intensity line plots of EGFP-MinD and streptavidin distribution of selections shown in the respective images. Scale bars: 50 µm. e Large-scale streptavidin gradient formation by MinDE is reversible. Representative images and kymograph (1) of a running MinDE assay in the presence of anchored streptavidin. Addition of sodium orthovanadate (Na 3 VO 4 ) leads to MinDE detachment which in turn leads to homogenization of streptavidin fluorescence on the membrane. Fluorescence intensity of streptavidin (cyan) and EGFP-MinD (green) is plotted over the duration of the time-lapse in the center (2) and at the rim of the MinDE spiral (3). Scale bars: 50 µm and 300 s. All experiments were performed independently three or more times under identical conditions

    Techniques Used: Concentration Assay, Binding Assay, Fluorescence, Homogenization

    21) Product Images from "The RNA-binding protein ILF3 binds to transposable element sequences in SINEUP lncRNAs"

    Article Title: The RNA-binding protein ILF3 binds to transposable element sequences in SINEUP lncRNAs

    Journal: The FASEB Journal

    doi: 10.1096/fj.201901618RR

    ILF3 binds the human FRAM in vitro and upon transfection of hminiSINEUP-GFP. A ) Schematic representation of hminiSINEUP-GFP constructs. The overlapping region with sense GFP mRNA, representing the BD (green), spans 39 nt of GFP 5′UTR (gray). The FRAM is the ED (red) of hSINEUP R12A-AS1 ( 30 ). hminiSINEUP-GFPΔFRAM presents the BD but lacks the FRAM sequence. B ) Analysis by phage ELISA of the binding of dsRBM2 to the human FRAM repeats RNA sequence. ELISA signals were normalized to the invSINEB2 of AS Uchl1 (SINEB2). As negative controls, bindings on streptavidin (strep) and 2 unrelated RNAs {polyuridine [poly(U)] and adenylate-uridylate–rich element (ARE)} were measured ( n = 3). C ) RNA-IP assay on endogenous ILF3 and ectopically expressed hminiSINEUP-GFP or hminiSINEUP-GFPΔFRAM in HEK 293T/17. IgGs were used as ILF3 immunoprecipitation (IP) specificity control. RNA enrichments in ILF3 IP fraction were quantified with real-time quantitative PCR and expressed as (2 Δ Ct ) × 100 ILF3 IP ÷ (2 Δ Ct ) × 100 IgG. Δ C t was calculated on input, and RNA content in IP or IgG was normalized on UBC mRNA. D ) ILF3 IP efficiency was checked by Western blot performed with anti-DRBP76 (ILF3) antibody. Data are representative of 4 independent experiments and indicate means ± sd . NS, not significant. * P
    Figure Legend Snippet: ILF3 binds the human FRAM in vitro and upon transfection of hminiSINEUP-GFP. A ) Schematic representation of hminiSINEUP-GFP constructs. The overlapping region with sense GFP mRNA, representing the BD (green), spans 39 nt of GFP 5′UTR (gray). The FRAM is the ED (red) of hSINEUP R12A-AS1 ( 30 ). hminiSINEUP-GFPΔFRAM presents the BD but lacks the FRAM sequence. B ) Analysis by phage ELISA of the binding of dsRBM2 to the human FRAM repeats RNA sequence. ELISA signals were normalized to the invSINEB2 of AS Uchl1 (SINEB2). As negative controls, bindings on streptavidin (strep) and 2 unrelated RNAs {polyuridine [poly(U)] and adenylate-uridylate–rich element (ARE)} were measured ( n = 3). C ) RNA-IP assay on endogenous ILF3 and ectopically expressed hminiSINEUP-GFP or hminiSINEUP-GFPΔFRAM in HEK 293T/17. IgGs were used as ILF3 immunoprecipitation (IP) specificity control. RNA enrichments in ILF3 IP fraction were quantified with real-time quantitative PCR and expressed as (2 Δ Ct ) × 100 ILF3 IP ÷ (2 Δ Ct ) × 100 IgG. Δ C t was calculated on input, and RNA content in IP or IgG was normalized on UBC mRNA. D ) ILF3 IP efficiency was checked by Western blot performed with anti-DRBP76 (ILF3) antibody. Data are representative of 4 independent experiments and indicate means ± sd . NS, not significant. * P

    Techniques Used: In Vitro, Transfection, Construct, Sequencing, Enzyme-linked Immunosorbent Assay, Binding Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction, Western Blot

    ILF3 is the dominant SINEUP-interacting ORF isolated by phage display selection. A ) Schematic representation of ILF3 domains: NF45-homology domain, nuclear localization signal (NLS), dsRBMs 1 and 2, RGG motif, and GQSY domain. B ) Reads alignment to ILF3 gene showed specific enrichment of dsRBM2 (black arrows) in invSINEB2 library (middle) and AS Uchl1 Δ5′ library (bottom) but not in the NS library (top). Blue bars indicate the gene; green bars correspond to exons. C ) Representative phage ELISA experiment of the binding of the invSINEB2 sequence to ILF3 and the RNA-recognition motif of negative controls (SRSF5 and hnRNPA3). D ) Analysis by phage ELISA of the binding of dsRBM2 to AS Uchl1 Δ5′ and invSINEB2 RNA sequences. E ) Analysis by GST ELISA of the binding specificity of ILF3 dsRBM1 and mouse-human dsRBM2 to AS Uchl1 Δ5′ and invSINEB2 RNA sequences. Domains were produced as GST fusion polypeptides. Strep, streptavidin. Data indicate means ± sd . Data are representative of n = 3 independent replicas.
    Figure Legend Snippet: ILF3 is the dominant SINEUP-interacting ORF isolated by phage display selection. A ) Schematic representation of ILF3 domains: NF45-homology domain, nuclear localization signal (NLS), dsRBMs 1 and 2, RGG motif, and GQSY domain. B ) Reads alignment to ILF3 gene showed specific enrichment of dsRBM2 (black arrows) in invSINEB2 library (middle) and AS Uchl1 Δ5′ library (bottom) but not in the NS library (top). Blue bars indicate the gene; green bars correspond to exons. C ) Representative phage ELISA experiment of the binding of the invSINEB2 sequence to ILF3 and the RNA-recognition motif of negative controls (SRSF5 and hnRNPA3). D ) Analysis by phage ELISA of the binding of dsRBM2 to AS Uchl1 Δ5′ and invSINEB2 RNA sequences. E ) Analysis by GST ELISA of the binding specificity of ILF3 dsRBM1 and mouse-human dsRBM2 to AS Uchl1 Δ5′ and invSINEB2 RNA sequences. Domains were produced as GST fusion polypeptides. Strep, streptavidin. Data indicate means ± sd . Data are representative of n = 3 independent replicas.

    Techniques Used: Isolation, Selection, Enzyme-linked Immunosorbent Assay, Binding Assay, Sequencing, Produced

    22) Product Images from ""Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation"

    Article Title: "Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation

    Journal: Analytical chemistry

    doi: 10.1021/ac301278s

    Flow distance traveled as a function of streptavidin concentration in biotin-modified microchannels (58 μm wide) of two different heights, with a 0.5 mm thick PDMS top layer. (A) 13 μm deep channels. (B) 17 μm deep channels.
    Figure Legend Snippet: Flow distance traveled as a function of streptavidin concentration in biotin-modified microchannels (58 μm wide) of two different heights, with a 0.5 mm thick PDMS top layer. (A) 13 μm deep channels. (B) 17 μm deep channels.

    Techniques Used: Flow Cytometry, Concentration Assay, Modification

    Effect of solution viscosity on the flow distance of 100 ng/mL (X) and 1.0 μg/mL (O) streptavidin solutions in biotin-modified 17 μm deep and 58 μm wide channels with a 0.5 mm PDMS top layer. Flow distance is only affected above
    Figure Legend Snippet: Effect of solution viscosity on the flow distance of 100 ng/mL (X) and 1.0 μg/mL (O) streptavidin solutions in biotin-modified 17 μm deep and 58 μm wide channels with a 0.5 mm PDMS top layer. Flow distance is only affected above

    Techniques Used: Flow Cytometry, Modification

    Effect of PDMS layer thickness on the flow distance for 1.0 μg/mL streptavidin in biotin-modified 17 μm deep and 58 μm wide channels. Flow distance decreases asymptotically as PDMS layer thickness is reduced.
    Figure Legend Snippet: Effect of PDMS layer thickness on the flow distance for 1.0 μg/mL streptavidin in biotin-modified 17 μm deep and 58 μm wide channels. Flow distance decreases asymptotically as PDMS layer thickness is reduced.

    Techniques Used: Flow Cytometry, Modification

    Plots of background subtracted, normalized fluorescence signal and peak area in 13 μm deep biotin-modified channels as a function of channel position and flow distance to probe constriction. (A–B) Unlabeled streptavidin (500 μg/mL)
    Figure Legend Snippet: Plots of background subtracted, normalized fluorescence signal and peak area in 13 μm deep biotin-modified channels as a function of channel position and flow distance to probe constriction. (A–B) Unlabeled streptavidin (500 μg/mL)

    Techniques Used: Fluorescence, Modification, Flow Cytometry

    23) Product Images from ""Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation"

    Article Title: "Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation

    Journal: Analytical chemistry

    doi: 10.1021/ac301278s

    Flow distance traveled as a function of streptavidin concentration in biotin-modified microchannels (58 μm wide) of two different heights, with a 0.5 mm thick PDMS top layer. (A) 13 μm deep channels. (B) 17 μm deep channels.
    Figure Legend Snippet: Flow distance traveled as a function of streptavidin concentration in biotin-modified microchannels (58 μm wide) of two different heights, with a 0.5 mm thick PDMS top layer. (A) 13 μm deep channels. (B) 17 μm deep channels.

    Techniques Used: Flow Cytometry, Concentration Assay, Modification

    Effect of solution viscosity on the flow distance of 100 ng/mL (X) and 1.0 μg/mL (O) streptavidin solutions in biotin-modified 17 μm deep and 58 μm wide channels with a 0.5 mm PDMS top layer. Flow distance is only affected above
    Figure Legend Snippet: Effect of solution viscosity on the flow distance of 100 ng/mL (X) and 1.0 μg/mL (O) streptavidin solutions in biotin-modified 17 μm deep and 58 μm wide channels with a 0.5 mm PDMS top layer. Flow distance is only affected above

    Techniques Used: Flow Cytometry, Modification

    Effect of PDMS layer thickness on the flow distance for 1.0 μg/mL streptavidin in biotin-modified 17 μm deep and 58 μm wide channels. Flow distance decreases asymptotically as PDMS layer thickness is reduced.
    Figure Legend Snippet: Effect of PDMS layer thickness on the flow distance for 1.0 μg/mL streptavidin in biotin-modified 17 μm deep and 58 μm wide channels. Flow distance decreases asymptotically as PDMS layer thickness is reduced.

    Techniques Used: Flow Cytometry, Modification

    Plots of background subtracted, normalized fluorescence signal and peak area in 13 μm deep biotin-modified channels as a function of channel position and flow distance to probe constriction. (A–B) Unlabeled streptavidin (500 μg/mL)
    Figure Legend Snippet: Plots of background subtracted, normalized fluorescence signal and peak area in 13 μm deep biotin-modified channels as a function of channel position and flow distance to probe constriction. (A–B) Unlabeled streptavidin (500 μg/mL)

    Techniques Used: Fluorescence, Modification, Flow Cytometry

    24) Product Images from "La-related protein 1 (LARP1) repression of TOP mRNA translation is mediated through its cap-binding domain and controlled by an adjacent regulatory region"

    Article Title: La-related protein 1 (LARP1) repression of TOP mRNA translation is mediated through its cap-binding domain and controlled by an adjacent regulatory region

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx1237

    The C-terminal half of LARP1 selectively binds TOP sequences and the adjacent cap structure. ( A ) LARP1 497–1019 selectively recognizes oligopyrimidine RNA sequences and the 5′ cap structure. Extracts were prepared from LARP1-null HEK-293T cells expressing either FLAG-tagged LARP1 497-1019 or an N-terminal fragment (1–496) and treated with vehicle (DMSO) or 250 nM Torin 1 for 2 h. Extracts were then incubated with TOP or non-TOP (nTOP) 10 nt RNAs that were either capped or uncapped and containing a 3′ biotin. RNAs were then isolated using streptavidin-coated beads and analyzed by western blotting for the indicated proteins. ( B ) Endogenous LARP1 selectively recognizes capped oligopyrimidine RNA sequences. Extracts were prepared from WT HEK-293T cells treated with DMSO or 250 nM Torin 1 for 2 h, and then incubated with TOP or non-TOP (nTOP) 10 nt RNA probes that were either capped or uncapped and contained a 3′ biotin. RNA probes were isolated as in (A) and analyzed by western blotting for the indicated proteins. ( C ) LARP1 497–1019 fails to interact with PABP. Extracts were prepared from LARP1-null HEK-293T cells expressing either FLAG-tagged LARP1 497-1019 or an N-terminal fragment (1–496) and treated with vehicle (DMSO) or 250 nM Torin 1 for 2 h. FLAG-tagged proteins were then isolated by FLAG-affinity purification in the presence of RNase A, and analyzed by western blotting for the indicated proteins. ( D ) LARP1 mutation that disrupts cap binding prevents TOP mRNA regulation. LARP1-null HEK-293T cells were transfected with the indicated LARP1 cDNAs, along with TOP and non-TOP (nTOP) reporters as in Figure 1D , treated with vehicle (DMSO) or 250 nM Torin 1 for 6 h, and then analyzed for levels of Renilla and firefly luciferase. Data are Renilla/firefly, normalized to vehicle-treated nTOP levels for each LARP1 construct ( n = 3, error bars are SD). ( E ) Expression levels of LARP1 497–1019 WT and Y883A fragments. Cell extracts from cells treated as in (D) were analyzed by western blotting for the indicated proteins.
    Figure Legend Snippet: The C-terminal half of LARP1 selectively binds TOP sequences and the adjacent cap structure. ( A ) LARP1 497–1019 selectively recognizes oligopyrimidine RNA sequences and the 5′ cap structure. Extracts were prepared from LARP1-null HEK-293T cells expressing either FLAG-tagged LARP1 497-1019 or an N-terminal fragment (1–496) and treated with vehicle (DMSO) or 250 nM Torin 1 for 2 h. Extracts were then incubated with TOP or non-TOP (nTOP) 10 nt RNAs that were either capped or uncapped and containing a 3′ biotin. RNAs were then isolated using streptavidin-coated beads and analyzed by western blotting for the indicated proteins. ( B ) Endogenous LARP1 selectively recognizes capped oligopyrimidine RNA sequences. Extracts were prepared from WT HEK-293T cells treated with DMSO or 250 nM Torin 1 for 2 h, and then incubated with TOP or non-TOP (nTOP) 10 nt RNA probes that were either capped or uncapped and contained a 3′ biotin. RNA probes were isolated as in (A) and analyzed by western blotting for the indicated proteins. ( C ) LARP1 497–1019 fails to interact with PABP. Extracts were prepared from LARP1-null HEK-293T cells expressing either FLAG-tagged LARP1 497-1019 or an N-terminal fragment (1–496) and treated with vehicle (DMSO) or 250 nM Torin 1 for 2 h. FLAG-tagged proteins were then isolated by FLAG-affinity purification in the presence of RNase A, and analyzed by western blotting for the indicated proteins. ( D ) LARP1 mutation that disrupts cap binding prevents TOP mRNA regulation. LARP1-null HEK-293T cells were transfected with the indicated LARP1 cDNAs, along with TOP and non-TOP (nTOP) reporters as in Figure 1D , treated with vehicle (DMSO) or 250 nM Torin 1 for 6 h, and then analyzed for levels of Renilla and firefly luciferase. Data are Renilla/firefly, normalized to vehicle-treated nTOP levels for each LARP1 construct ( n = 3, error bars are SD). ( E ) Expression levels of LARP1 497–1019 WT and Y883A fragments. Cell extracts from cells treated as in (D) were analyzed by western blotting for the indicated proteins.

    Techniques Used: Expressing, Incubation, Isolation, Western Blot, Affinity Purification, Mutagenesis, Binding Assay, Transfection, Luciferase, Construct

    25) Product Images from "The PriA Replication Restart Protein Blocks Replicase Access Prior to Helicase Assembly and Directs Template Specificity through Its ATPase Activity *"

    Article Title: The PriA Replication Restart Protein Blocks Replicase Access Prior to Helicase Assembly and Directs Template Specificity through Its ATPase Activity *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.435966

    PriA and holoenzyme do not coexist on PriA-inhibited replication forks. A , diagram depicting two possible models of PriA inhibiting the strand displacement reaction and the expected result of each for reactions on streptavidin ( SA ) beads. The primer is labeled on the 5′-end with 32 P so that primer extension and helicase activity can be monitored. The primer contains a biotin near the 5′-end so that substrates can be conjugated to streptavidin-linked beads. Scheme 1 depicts PriA blocking the 3′-OH of the primer, physically preventing Pol III HE from binding. Scheme 2 portrays an inhibition model where both PriA and Pol III HE bind to the substrate. Primosomal proteins (PriB, DnaT, DnaB, and DnaC) were added as described under “Experimental Procedures.” B , denaturing gel analysis to monitor primer extension by E. coli Pol III (exo-). Lanes 11–13 are dilutions of the positive control lane 3 to establish detection limits. For both B and C , lanes 8 and 9 contain the full Pol III HE but in lane 8 , the β-subunit was omitted, and in lane 9 , SSB was omitted. C , native gel analysis to monitor substrate unwinding by E. coli DnaB helicase. The upper band is the replication fork, and the 90/90 duplex product in those cases is where replication occurs. The lower band is the displaced leading strand primer template. In lane 5 , ∼45% of the substrate was unwound by the helicase. In lane 7 , ∼40% of the substrate was unwound by the helicase. In all other lanes, the amount of substrate unwound is not significantly above background. D , denaturing gel analysis to monitor primer extension by E. coli Pol III (exo-) on immobilized substrate without a washing step. Experiment carried out as described under “Experimental Procedures,” except after incubation with Pol III HE components, the washing steps were omitted. In lane 5 , ∼45% of the primer is elongated.
    Figure Legend Snippet: PriA and holoenzyme do not coexist on PriA-inhibited replication forks. A , diagram depicting two possible models of PriA inhibiting the strand displacement reaction and the expected result of each for reactions on streptavidin ( SA ) beads. The primer is labeled on the 5′-end with 32 P so that primer extension and helicase activity can be monitored. The primer contains a biotin near the 5′-end so that substrates can be conjugated to streptavidin-linked beads. Scheme 1 depicts PriA blocking the 3′-OH of the primer, physically preventing Pol III HE from binding. Scheme 2 portrays an inhibition model where both PriA and Pol III HE bind to the substrate. Primosomal proteins (PriB, DnaT, DnaB, and DnaC) were added as described under “Experimental Procedures.” B , denaturing gel analysis to monitor primer extension by E. coli Pol III (exo-). Lanes 11–13 are dilutions of the positive control lane 3 to establish detection limits. For both B and C , lanes 8 and 9 contain the full Pol III HE but in lane 8 , the β-subunit was omitted, and in lane 9 , SSB was omitted. C , native gel analysis to monitor substrate unwinding by E. coli DnaB helicase. The upper band is the replication fork, and the 90/90 duplex product in those cases is where replication occurs. The lower band is the displaced leading strand primer template. In lane 5 , ∼45% of the substrate was unwound by the helicase. In lane 7 , ∼40% of the substrate was unwound by the helicase. In all other lanes, the amount of substrate unwound is not significantly above background. D , denaturing gel analysis to monitor primer extension by E. coli Pol III (exo-) on immobilized substrate without a washing step. Experiment carried out as described under “Experimental Procedures,” except after incubation with Pol III HE components, the washing steps were omitted. In lane 5 , ∼45% of the primer is elongated.

    Techniques Used: Labeling, Activity Assay, Blocking Assay, Binding Assay, Inhibition, Positive Control, Incubation

    26) Product Images from "MOrPH-PhD: An Integrated Phage Display Platform for the Discovery of Functional Genetically Encoded Peptide Macrocycles"

    Article Title: MOrPH-PhD: An Integrated Phage Display Platform for the Discovery of Functional Genetically Encoded Peptide Macrocycles

    Journal: ACS Central Science

    doi: 10.1021/acscentsci.9b00927

    Display of O2beY-containing peptide on M13 phages. (A) Incorporation of cysteine-reactive O2beY and cysteine-unreactive OpgY into a linear nonapeptide (NB9) N-terminally fused to the M13 phage coat protein pIII. (B) Plaque forming units (p.f.u.) generated in the absence and presence of either noncanonical amino acid from E. coli cells expressing the polyspecific O2beY-RS synthetase, as determined by the phage titer assay. (C) Selective recovery of O2beY-displaying phages over OpgY-displaying ones using streptavidin-coated beads after phage exposure to biotin-conjugated cysteine reagent (Biot-Cys).
    Figure Legend Snippet: Display of O2beY-containing peptide on M13 phages. (A) Incorporation of cysteine-reactive O2beY and cysteine-unreactive OpgY into a linear nonapeptide (NB9) N-terminally fused to the M13 phage coat protein pIII. (B) Plaque forming units (p.f.u.) generated in the absence and presence of either noncanonical amino acid from E. coli cells expressing the polyspecific O2beY-RS synthetase, as determined by the phage titer assay. (C) Selective recovery of O2beY-displaying phages over OpgY-displaying ones using streptavidin-coated beads after phage exposure to biotin-conjugated cysteine reagent (Biot-Cys).

    Techniques Used: Generated, Expressing, Titer Assay

    Affinity selection of streptavidin-binding peptide macrocycles. (A) Libraries of semirandomized O2beY-linked peptide macrocycles (X = NNK codon) displayed on phages.(B) Hit sequences identified by deep sequencing (relative abundance = n /54 000 sequences) after library panning against streptavidin-coated magnetic beads. K D values refer to the corresponding FLAG-macrocycle-CBD constructs in purified form. (C) Binding curves for selected peptide macrocycle hits as determined using a direct binding assay with plate-immobilized streptavidin and HRP-conjugated anti-FLAG antibody for detection of the bound peptide. CBD alone shows no detectable binding to streptavidin. (D) Phage enrichment over the four rounds of affinity selection and amplification as determined via the phage titer assay.
    Figure Legend Snippet: Affinity selection of streptavidin-binding peptide macrocycles. (A) Libraries of semirandomized O2beY-linked peptide macrocycles (X = NNK codon) displayed on phages.(B) Hit sequences identified by deep sequencing (relative abundance = n /54 000 sequences) after library panning against streptavidin-coated magnetic beads. K D values refer to the corresponding FLAG-macrocycle-CBD constructs in purified form. (C) Binding curves for selected peptide macrocycle hits as determined using a direct binding assay with plate-immobilized streptavidin and HRP-conjugated anti-FLAG antibody for detection of the bound peptide. CBD alone shows no detectable binding to streptavidin. (D) Phage enrichment over the four rounds of affinity selection and amplification as determined via the phage titer assay.

    Techniques Used: Selection, Binding Assay, Sequencing, Magnetic Beads, Construct, Purification, Amplification, Titer Assay

    27) Product Images from "Rapid Affinity Immunochromatography Column-Based Tests for Sensitive Detection of Clostridium botulinum Neurotoxins and Escherichia coli O157 ▿"

    Article Title: Rapid Affinity Immunochromatography Column-Based Tests for Sensitive Detection of Clostridium botulinum Neurotoxins and Escherichia coli O157 ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.03059-09

    Diagram of the assay. A test sample is preincubated with a biotinylated detection antibody for 10 min, resulting in formation of an antibody-antigen complex. The antibody-coated filter captures the antigen-antibody complex from solution, while unbound antibody flows through. Then a second solution, which contains HRP-labeled streptavidin that binds to the biotin, is added. Finally, an insoluble substrate for the HRP enzyme is added, which produces a visible band following precipitation of TMB where the HRP, and thus the antigen, is present. For the E. coli O157 assay, unbound cells were run through the column to bind to the capture antibody before addition of the detection antibody.
    Figure Legend Snippet: Diagram of the assay. A test sample is preincubated with a biotinylated detection antibody for 10 min, resulting in formation of an antibody-antigen complex. The antibody-coated filter captures the antigen-antibody complex from solution, while unbound antibody flows through. Then a second solution, which contains HRP-labeled streptavidin that binds to the biotin, is added. Finally, an insoluble substrate for the HRP enzyme is added, which produces a visible band following precipitation of TMB where the HRP, and thus the antigen, is present. For the E. coli O157 assay, unbound cells were run through the column to bind to the capture antibody before addition of the detection antibody.

    Techniques Used: Labeling

    28) Product Images from "The homophilic receptor PTPRK selectively dephosphorylates multiple junctional regulators to promote cell–cell adhesion"

    Article Title: The homophilic receptor PTPRK selectively dephosphorylates multiple junctional regulators to promote cell–cell adhesion

    Journal: eLife

    doi: 10.7554/eLife.44597

    In vitro dephosphorylation assays and generation of RPTP chimeras. ( A ) The indicated PTPRK and PTPRM domains were assayed for phosphatase activity using the pNPP colorimetric assay. Control wells contained pNPP only. Protein amounts used are shown. ( B ) Pervanadate-treated MCF10A lysates were incubated with predetermined amounts of the indicated domains to give equal phosphatase-activity, prior to phosphotyrosine immunoprecipitation and immunoblot analysis. ( C ) Recombinant proteins consisting of combinations of PTPRK and PTPRM D1 and D2 domains were expressed in and using Ni-NTA affinity resin. Purified proteins were then subjected to size exclusion chromatography. ( D ) Recombinant His- and Avi-tagged PTPRK and PTPRM chimeric domains were purified from E. coli cultured in biotin-supplemented media, incubated ±streptavidin and subjected to SDS-PAGE and Coomassie staining, to determine the extent of biotinylation. Arrows indicate the purified domains and the respective streptavidin-induced mobility shift. ( E ) The indicated recombinant PTPRK and PTPRM chimeric domains were incubated were assayed for phosphatase activity using the pNPP colorimetric assay. Control wells contained pNPP. Protein amounts used are shown.
    Figure Legend Snippet: In vitro dephosphorylation assays and generation of RPTP chimeras. ( A ) The indicated PTPRK and PTPRM domains were assayed for phosphatase activity using the pNPP colorimetric assay. Control wells contained pNPP only. Protein amounts used are shown. ( B ) Pervanadate-treated MCF10A lysates were incubated with predetermined amounts of the indicated domains to give equal phosphatase-activity, prior to phosphotyrosine immunoprecipitation and immunoblot analysis. ( C ) Recombinant proteins consisting of combinations of PTPRK and PTPRM D1 and D2 domains were expressed in and using Ni-NTA affinity resin. Purified proteins were then subjected to size exclusion chromatography. ( D ) Recombinant His- and Avi-tagged PTPRK and PTPRM chimeric domains were purified from E. coli cultured in biotin-supplemented media, incubated ±streptavidin and subjected to SDS-PAGE and Coomassie staining, to determine the extent of biotinylation. Arrows indicate the purified domains and the respective streptavidin-induced mobility shift. ( E ) The indicated recombinant PTPRK and PTPRM chimeric domains were incubated were assayed for phosphatase activity using the pNPP colorimetric assay. Control wells contained pNPP. Protein amounts used are shown.

    Techniques Used: In Vitro, De-Phosphorylation Assay, Activity Assay, Colorimetric Assay, Incubation, Immunoprecipitation, Recombinant, Purification, Size-exclusion Chromatography, Cell Culture, SDS Page, Staining, Mobility Shift

    Purification of biotinylated recombinant PTPRK domains. ( A ) His- and Avi-tagged PTPRK domains were expressed in E. coli cultured in biotin-supplemented media and purified using Nickel-NTA beads, followed by size exclusion chromatography (SEC). DA = D1057A mutant. CS = C1089S mutant. ( B ) SEC-purified proteins bound to streptavidin resin were eluted and resolved by SDS-PAGE followed by Coomassie staining. In; input, B; beads. ( C ) The phosphatase activity of indicated amounts of purified proteins was assessed using the Biomol green assay with two tyrosine phosphorylated peptides as substrates and was quantified at 620 nm. ( D ) Recombinant proteins bound to streptavidin resin were used in pull down assays from pervanadate treated Hs27 fibroblast lysates. After extensive washing, bound proteins were eluted in sample buffer and analyzed by immunoblot.
    Figure Legend Snippet: Purification of biotinylated recombinant PTPRK domains. ( A ) His- and Avi-tagged PTPRK domains were expressed in E. coli cultured in biotin-supplemented media and purified using Nickel-NTA beads, followed by size exclusion chromatography (SEC). DA = D1057A mutant. CS = C1089S mutant. ( B ) SEC-purified proteins bound to streptavidin resin were eluted and resolved by SDS-PAGE followed by Coomassie staining. In; input, B; beads. ( C ) The phosphatase activity of indicated amounts of purified proteins was assessed using the Biomol green assay with two tyrosine phosphorylated peptides as substrates and was quantified at 620 nm. ( D ) Recombinant proteins bound to streptavidin resin were used in pull down assays from pervanadate treated Hs27 fibroblast lysates. After extensive washing, bound proteins were eluted in sample buffer and analyzed by immunoblot.

    Techniques Used: Purification, Recombinant, Cell Culture, Size-exclusion Chromatography, Mutagenesis, SDS Page, Staining, Activity Assay

    The PTPRK-dependent tyrosine phosphoproteome. ( A ) After SEC, proteins were incubated with or without streptavidin and subjected to SDS PAGE followed by Coomassie staining to determine the extent of biotinylation. Arrows indicate the purified domains and the respective streptavidin-induced mobility shift. ( B ) Volcano plot of tyrosine phosphosites detected in PTPRK KO and wildtype MCF10As. Phosphosites > 50% enriched in (p
    Figure Legend Snippet: The PTPRK-dependent tyrosine phosphoproteome. ( A ) After SEC, proteins were incubated with or without streptavidin and subjected to SDS PAGE followed by Coomassie staining to determine the extent of biotinylation. Arrows indicate the purified domains and the respective streptavidin-induced mobility shift. ( B ) Volcano plot of tyrosine phosphosites detected in PTPRK KO and wildtype MCF10As. Phosphosites > 50% enriched in (p

    Techniques Used: Size-exclusion Chromatography, Incubation, SDS Page, Staining, Purification, Mobility Shift

    29) Product Images from "A Novel mRNA Level Subtraction Method for Quick Identification of Target-Orientated Uniquely Expressed Genes Between Peanut Immature Pod and Leaf"

    Article Title: A Novel mRNA Level Subtraction Method for Quick Identification of Target-Orientated Uniquely Expressed Genes Between Peanut Immature Pod and Leaf

    Journal: Biological Procedures Online

    doi: 10.1007/s12575-009-9022-z

    General scheme applied for identifying peanut immature pod-specific genes (tracer mRNA (1)) after a single round subtraction . B biotin, S streptavidin, M magnetic bead.
    Figure Legend Snippet: General scheme applied for identifying peanut immature pod-specific genes (tracer mRNA (1)) after a single round subtraction . B biotin, S streptavidin, M magnetic bead.

    Techniques Used:

    30) Product Images from "Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes *"

    Article Title: Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.730135

    NPH I translocates 5′ to 3′ on single-stranded DNA. A , phosphorimage of a 10% native polyacrylamide gel of a streptavidin displacement assay. Assays were conducted on 36-mer oligonucleotides that were biotinylated at either the 3′
    Figure Legend Snippet: NPH I translocates 5′ to 3′ on single-stranded DNA. A , phosphorimage of a 10% native polyacrylamide gel of a streptavidin displacement assay. Assays were conducted on 36-mer oligonucleotides that were biotinylated at either the 3′

    Techniques Used:

    31) Product Images from "A method for the isolation and characterization of functional murine monoclonal antibodies by single B cell cloning"

    Article Title: A method for the isolation and characterization of functional murine monoclonal antibodies by single B cell cloning

    Journal: Journal of immunological methods

    doi: 10.1016/j.jim.2017.05.010

    Workflow summary of mAb discovery pipeline in mice. Upon completion of the immunization regimen, spleens are harvested and gently homogenized to a single-cell suspension. The cell suspension is treated by negative magnetically-assisted selection until predominantly B cells remain. The B-cell suspension is stained using a cocktail of antibodies and Ag-streptavidin (as well as decoy-streptavidin) complexes to facilitate the sorting of Ag-positive class-switched cells. The cells are then cultured and induced to express antibodies in presence of supportive cytokines and feeder cells. Culture-conditioned supernatants containing target antibodies are then screened using Ag-specific ELISA, and positive wells are harvested for RNA isolation. Ab-encoding RNA is then reverse-transcribed, amplified and cloned into an expression cassette enabling recombinant production of the mAbs, followed by functional validation.
    Figure Legend Snippet: Workflow summary of mAb discovery pipeline in mice. Upon completion of the immunization regimen, spleens are harvested and gently homogenized to a single-cell suspension. The cell suspension is treated by negative magnetically-assisted selection until predominantly B cells remain. The B-cell suspension is stained using a cocktail of antibodies and Ag-streptavidin (as well as decoy-streptavidin) complexes to facilitate the sorting of Ag-positive class-switched cells. The cells are then cultured and induced to express antibodies in presence of supportive cytokines and feeder cells. Culture-conditioned supernatants containing target antibodies are then screened using Ag-specific ELISA, and positive wells are harvested for RNA isolation. Ab-encoding RNA is then reverse-transcribed, amplified and cloned into an expression cassette enabling recombinant production of the mAbs, followed by functional validation.

    Techniques Used: Mouse Assay, Selection, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Isolation, Amplification, Clone Assay, Expressing, Recombinant, Functional Assay

    32) Product Images from ""Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation"

    Article Title: "Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation

    Journal: Analytical chemistry

    doi: 10.1021/ac301278s

    Flow distance traveled as a function of streptavidin concentration in biotin-modified microchannels (58 μm wide) of two different heights, with a 0.5 mm thick PDMS top layer. (A) 13 μm deep channels. (B) 17 μm deep channels.
    Figure Legend Snippet: Flow distance traveled as a function of streptavidin concentration in biotin-modified microchannels (58 μm wide) of two different heights, with a 0.5 mm thick PDMS top layer. (A) 13 μm deep channels. (B) 17 μm deep channels.

    Techniques Used: Flow Cytometry, Concentration Assay, Modification

    Effect of solution viscosity on the flow distance of 100 ng/mL (X) and 1.0 μg/mL (O) streptavidin solutions in biotin-modified 17 μm deep and 58 μm wide channels with a 0.5 mm PDMS top layer. Flow distance is only affected above
    Figure Legend Snippet: Effect of solution viscosity on the flow distance of 100 ng/mL (X) and 1.0 μg/mL (O) streptavidin solutions in biotin-modified 17 μm deep and 58 μm wide channels with a 0.5 mm PDMS top layer. Flow distance is only affected above

    Techniques Used: Flow Cytometry, Modification

    Effect of PDMS layer thickness on the flow distance for 1.0 μg/mL streptavidin in biotin-modified 17 μm deep and 58 μm wide channels. Flow distance decreases asymptotically as PDMS layer thickness is reduced.
    Figure Legend Snippet: Effect of PDMS layer thickness on the flow distance for 1.0 μg/mL streptavidin in biotin-modified 17 μm deep and 58 μm wide channels. Flow distance decreases asymptotically as PDMS layer thickness is reduced.

    Techniques Used: Flow Cytometry, Modification

    Plots of background subtracted, normalized fluorescence signal and peak area in 13 μm deep biotin-modified channels as a function of channel position and flow distance to probe constriction. (A–B) Unlabeled streptavidin (500 μg/mL)
    Figure Legend Snippet: Plots of background subtracted, normalized fluorescence signal and peak area in 13 μm deep biotin-modified channels as a function of channel position and flow distance to probe constriction. (A–B) Unlabeled streptavidin (500 μg/mL)

    Techniques Used: Fluorescence, Modification, Flow Cytometry

    33) Product Images from "MOrPH-PhD: An Integrated Phage Display Platform for the Discovery of Functional Genetically Encoded Peptide Macrocycles"

    Article Title: MOrPH-PhD: An Integrated Phage Display Platform for the Discovery of Functional Genetically Encoded Peptide Macrocycles

    Journal: ACS Central Science

    doi: 10.1021/acscentsci.9b00927

    Affinity selection of streptavidin-binding peptide macrocycles. (A) Libraries of semirandomized O2beY-linked peptide macrocycles (X = NNK codon) displayed on phages.(B) Hit sequences identified by deep sequencing (relative abundance = n /54 000 sequences) after library panning against streptavidin-coated magnetic beads. K D values refer to the corresponding FLAG-macrocycle-CBD constructs in purified form. (C) Binding curves for selected peptide macrocycle hits as determined using a direct binding assay with plate-immobilized streptavidin and HRP-conjugated anti-FLAG antibody for detection of the bound peptide. CBD alone shows no detectable binding to streptavidin. (D) Phage enrichment over the four rounds of affinity selection and amplification as determined via the phage titer assay.
    Figure Legend Snippet: Affinity selection of streptavidin-binding peptide macrocycles. (A) Libraries of semirandomized O2beY-linked peptide macrocycles (X = NNK codon) displayed on phages.(B) Hit sequences identified by deep sequencing (relative abundance = n /54 000 sequences) after library panning against streptavidin-coated magnetic beads. K D values refer to the corresponding FLAG-macrocycle-CBD constructs in purified form. (C) Binding curves for selected peptide macrocycle hits as determined using a direct binding assay with plate-immobilized streptavidin and HRP-conjugated anti-FLAG antibody for detection of the bound peptide. CBD alone shows no detectable binding to streptavidin. (D) Phage enrichment over the four rounds of affinity selection and amplification as determined via the phage titer assay.

    Techniques Used: Selection, Binding Assay, Sequencing, Magnetic Beads, Construct, Purification, Amplification, Titer Assay

    34) Product Images from "Identification of sialylated glycoproteins from metabolically oligosaccharide engineered pancreatic cells"

    Article Title: Identification of sialylated glycoproteins from metabolically oligosaccharide engineered pancreatic cells

    Journal: Clinical Proteomics

    doi: 10.1186/s12014-015-9083-8

    Strategy for analyzing azide-modified sialoglycoproteins. The strategy used to analyze the samples includes multiple steps as follows: 1) Cells were metabolically labeled using 1,3,4- O -Bu 3 ManNAz. 2) Proteins were extracted using RIPA buffer at which point samples were divided with one set of aliquots used for steps 4 and 5 and another set of aliquots used for steps 6 through 9. 3) Azide-labeled proteins were biotinylated using through the Staudinger reaction using biotin-PEG 3 -phosphine and excess reagent was removed by protein precipitation. 4) Biotinlabeled, azide-modified proteins were purified using monomeric avidin agarose. 5) Glycan profiles of biotin-labeled, azide-modified proteins were determined by lectin microarray analysis. 6) Proteins were trypsin digested after biotinylation. 7) Biotin-labeled peptides were coupled to streptavidin agarose. 8) PNGase F was used to release the formerly N -glycosylated peptide from the agarose beads. 9) The released peptides were analyzed by LC-MS.
    Figure Legend Snippet: Strategy for analyzing azide-modified sialoglycoproteins. The strategy used to analyze the samples includes multiple steps as follows: 1) Cells were metabolically labeled using 1,3,4- O -Bu 3 ManNAz. 2) Proteins were extracted using RIPA buffer at which point samples were divided with one set of aliquots used for steps 4 and 5 and another set of aliquots used for steps 6 through 9. 3) Azide-labeled proteins were biotinylated using through the Staudinger reaction using biotin-PEG 3 -phosphine and excess reagent was removed by protein precipitation. 4) Biotinlabeled, azide-modified proteins were purified using monomeric avidin agarose. 5) Glycan profiles of biotin-labeled, azide-modified proteins were determined by lectin microarray analysis. 6) Proteins were trypsin digested after biotinylation. 7) Biotin-labeled peptides were coupled to streptavidin agarose. 8) PNGase F was used to release the formerly N -glycosylated peptide from the agarose beads. 9) The released peptides were analyzed by LC-MS.

    Techniques Used: Modification, Metabolic Labelling, Labeling, Purification, Avidin-Biotin Assay, Microarray, Liquid Chromatography with Mass Spectroscopy

    35) Product Images from "A role for 2-Cys peroxiredoxins in facilitating cytosolic protein thiol oxidation"

    Article Title: A role for 2-Cys peroxiredoxins in facilitating cytosolic protein thiol oxidation

    Journal: Nature chemical biology

    doi: 10.1038/nchembio.2536

    Trx1 and Trxr1 are not required for transmission of oxidative equivalents to cytosolic proteins. ( a , b ) HAP1 cells proficient (WT) or deficient (ΔPrx1+2) in Prx1+2 expression ( a ) and HEK293T cells induced to express scrambled (SCR) or specific (Prx1+2) shRNA ( b ) were treated for 1 h with 10 μM ( a ) or 20 μM ( b ) auranofin or solvent control (DMSO), or were left untreated (UT). Overall protein thiol oxidation was assessed by kinetic trapping and analyzed by immunoblotting (IB). ( c ) HEK293T cells induced to express scrambled or specific (Prx1+2) shRNA were transfected with scrambled or specific (Trx1) siRNA and exposed to the indicated concentrations of H 2 O 2 . NR, nonreducing; R, reducing conditions; SBP, streptavidin binding peptide; Trx-Trap, eluate from Trx trapping beads. Blots are representative of ≥ 3 independent experiments.
    Figure Legend Snippet: Trx1 and Trxr1 are not required for transmission of oxidative equivalents to cytosolic proteins. ( a , b ) HAP1 cells proficient (WT) or deficient (ΔPrx1+2) in Prx1+2 expression ( a ) and HEK293T cells induced to express scrambled (SCR) or specific (Prx1+2) shRNA ( b ) were treated for 1 h with 10 μM ( a ) or 20 μM ( b ) auranofin or solvent control (DMSO), or were left untreated (UT). Overall protein thiol oxidation was assessed by kinetic trapping and analyzed by immunoblotting (IB). ( c ) HEK293T cells induced to express scrambled or specific (Prx1+2) shRNA were transfected with scrambled or specific (Trx1) siRNA and exposed to the indicated concentrations of H 2 O 2 . NR, nonreducing; R, reducing conditions; SBP, streptavidin binding peptide; Trx-Trap, eluate from Trx trapping beads. Blots are representative of ≥ 3 independent experiments.

    Techniques Used: Transmission Assay, Expressing, shRNA, Transfection, Binding Assay

    H 2 O 2 -induced cytosolic protein thiol oxidation depends on cytosolic peroxiredoxins. ( a ) Scheme depicting theoretical possibilities for H 2 O 2 -derived oxidizing equivalents to reach and oxidize redox-regulated proteins. Left column, canonical flow of oxidizing equivalents from H 2 O 2 to NADPH through Prx1/2, Trx1 and TrxR1. Middle column, branch points (i–iv) potentially involved in the delivery of oxidizing equivalents to redox-regulated target proteins. Right column, reduction of oxidized target proteins by the thioredoxin system. All molecules are shown in the oxidized state. ( b ) Scheme depicting the mechanism-based kinetic trapping approach used to monitor cytosolic protein thiol oxidation. Prior to cell lysis, free thiols are blocked with N -ethylmaleimide (NEM; black squares). Cells are osmotically disrupted to release cytosolic proteins. Disulfide-containing proteins are selectively captured by the immobilized recombinant trapping mutant of human Trx1. ( c , d ) HAP1 cells proficient (wild type, WT) or deficient (ΔPrx1+2) in Prx1+2 expression ( c ) and HEK293T cells induced to express either scrambled (SCR) or specific (Prx1+2) shRNA ( d ) were exposed to the indicated concentrations of H 2 O 2 for 15 s. Overall protein thiol oxidation, reflected by the formation of Trx-S-S-X conjugates, was assessed by the kinetic trapping approach and analyzed by immunoblotting against the SBP tag of the Trx1 trapping mutant. In c and d , different types of Trx conjugates are indicated. The Trx-S-S-Prx and Trx-S-S-(Prx) 2 ). Uncropped blots for c and d . IB, immunoblotting; NR, nonreducing; R, reducing conditions; SBP, streptavidin-binding peptide; Trx-Trap, eluate from Trx trapping beads. Blots are representative of ≥3 independent experiments.
    Figure Legend Snippet: H 2 O 2 -induced cytosolic protein thiol oxidation depends on cytosolic peroxiredoxins. ( a ) Scheme depicting theoretical possibilities for H 2 O 2 -derived oxidizing equivalents to reach and oxidize redox-regulated proteins. Left column, canonical flow of oxidizing equivalents from H 2 O 2 to NADPH through Prx1/2, Trx1 and TrxR1. Middle column, branch points (i–iv) potentially involved in the delivery of oxidizing equivalents to redox-regulated target proteins. Right column, reduction of oxidized target proteins by the thioredoxin system. All molecules are shown in the oxidized state. ( b ) Scheme depicting the mechanism-based kinetic trapping approach used to monitor cytosolic protein thiol oxidation. Prior to cell lysis, free thiols are blocked with N -ethylmaleimide (NEM; black squares). Cells are osmotically disrupted to release cytosolic proteins. Disulfide-containing proteins are selectively captured by the immobilized recombinant trapping mutant of human Trx1. ( c , d ) HAP1 cells proficient (wild type, WT) or deficient (ΔPrx1+2) in Prx1+2 expression ( c ) and HEK293T cells induced to express either scrambled (SCR) or specific (Prx1+2) shRNA ( d ) were exposed to the indicated concentrations of H 2 O 2 for 15 s. Overall protein thiol oxidation, reflected by the formation of Trx-S-S-X conjugates, was assessed by the kinetic trapping approach and analyzed by immunoblotting against the SBP tag of the Trx1 trapping mutant. In c and d , different types of Trx conjugates are indicated. The Trx-S-S-Prx and Trx-S-S-(Prx) 2 ). Uncropped blots for c and d . IB, immunoblotting; NR, nonreducing; R, reducing conditions; SBP, streptavidin-binding peptide; Trx-Trap, eluate from Trx trapping beads. Blots are representative of ≥3 independent experiments.

    Techniques Used: Derivative Assay, Flow Cytometry, Lysis, Recombinant, Mutagenesis, Expressing, shRNA, Binding Assay

    Upon H 2 O 2 exposure, peroxiredoxins form transient disulfide exchange intermediates with other proteins. ( a ) HEK293T cells expressing SBP-tagged Prx family members (1, 2, 3, 5 and 6) were exposed to 100 μM H 2 O 2 for 1 min (+) or left untreated (−). Free thiols were blocked by NEM before cell lysis. Mixed disulfide intermediates (outlined by dashed-line rectangles) were visualized on nonreducing gels by immunoblotting against the streptavidin binding peptide (SBP) tag. The blot is representative of three independent experiments. EV, empty vector. ( b ) Scheme explaining the pattern of proteins on two-dimensional nonreducing/reducing diagonal gels. *, interaction partners (X) released from X-S-S-Prx conjugates; **, interaction partners (X) released from X-S-S-(Prx) 2 conjugates; ***, Prx-SBP or Prx (end.) released from X-S-S-Prx or X-S-S-(Prx) 2 conjugates; #, Prx-SBP or Prx (end.) that was originally in the monomeric form; ##, Prx-SBP or Prx (end.) that was originally in the dimeric form; end., endogenous. Red and blue marks indicate molecular weight markers. ( c , d ) 2 × 10 8 HEK293T cells expressing either wild-type (WT), resolving cysteine-deficient (ΔC R ) or double (resolving and peroxidatic) cysteine-deficient (ΔC P ΔC R ) Prx1-SBP ( c ) or Prx2-SBP ( d ) were exposed to 100 μM H 2 O 2 for 3 min. Following thiol blocking and affinity purification, covalent interactions were analyzed by two-dimensional nonreducing/reducing diagonal SDS–PAGE. In c , the cartoons illustrate the nature of the disulfide linked complexes that lead to the formation of the two lower diagonals. In c and d , the arrows indicate the direction of protein migration.
    Figure Legend Snippet: Upon H 2 O 2 exposure, peroxiredoxins form transient disulfide exchange intermediates with other proteins. ( a ) HEK293T cells expressing SBP-tagged Prx family members (1, 2, 3, 5 and 6) were exposed to 100 μM H 2 O 2 for 1 min (+) or left untreated (−). Free thiols were blocked by NEM before cell lysis. Mixed disulfide intermediates (outlined by dashed-line rectangles) were visualized on nonreducing gels by immunoblotting against the streptavidin binding peptide (SBP) tag. The blot is representative of three independent experiments. EV, empty vector. ( b ) Scheme explaining the pattern of proteins on two-dimensional nonreducing/reducing diagonal gels. *, interaction partners (X) released from X-S-S-Prx conjugates; **, interaction partners (X) released from X-S-S-(Prx) 2 conjugates; ***, Prx-SBP or Prx (end.) released from X-S-S-Prx or X-S-S-(Prx) 2 conjugates; #, Prx-SBP or Prx (end.) that was originally in the monomeric form; ##, Prx-SBP or Prx (end.) that was originally in the dimeric form; end., endogenous. Red and blue marks indicate molecular weight markers. ( c , d ) 2 × 10 8 HEK293T cells expressing either wild-type (WT), resolving cysteine-deficient (ΔC R ) or double (resolving and peroxidatic) cysteine-deficient (ΔC P ΔC R ) Prx1-SBP ( c ) or Prx2-SBP ( d ) were exposed to 100 μM H 2 O 2 for 3 min. Following thiol blocking and affinity purification, covalent interactions were analyzed by two-dimensional nonreducing/reducing diagonal SDS–PAGE. In c , the cartoons illustrate the nature of the disulfide linked complexes that lead to the formation of the two lower diagonals. In c and d , the arrows indicate the direction of protein migration.

    Techniques Used: Expressing, Lysis, Binding Assay, Plasmid Preparation, Molecular Weight, Blocking Assay, Affinity Purification, SDS Page, Migration

    36) Product Images from ""Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation"

    Article Title: "Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation

    Journal: Analytical chemistry

    doi: 10.1021/ac301278s

    Flow distance traveled as a function of streptavidin concentration in biotin-modified microchannels (58 μm wide) of two different heights, with a 0.5 mm thick PDMS top layer. (A) 13 μm deep channels. (B) 17 μm deep channels.
    Figure Legend Snippet: Flow distance traveled as a function of streptavidin concentration in biotin-modified microchannels (58 μm wide) of two different heights, with a 0.5 mm thick PDMS top layer. (A) 13 μm deep channels. (B) 17 μm deep channels.

    Techniques Used: Flow Cytometry, Concentration Assay, Modification

    Effect of solution viscosity on the flow distance of 100 ng/mL (X) and 1.0 μg/mL (O) streptavidin solutions in biotin-modified 17 μm deep and 58 μm wide channels with a 0.5 mm PDMS top layer. Flow distance is only affected above
    Figure Legend Snippet: Effect of solution viscosity on the flow distance of 100 ng/mL (X) and 1.0 μg/mL (O) streptavidin solutions in biotin-modified 17 μm deep and 58 μm wide channels with a 0.5 mm PDMS top layer. Flow distance is only affected above

    Techniques Used: Flow Cytometry, Modification

    Effect of PDMS layer thickness on the flow distance for 1.0 μg/mL streptavidin in biotin-modified 17 μm deep and 58 μm wide channels. Flow distance decreases asymptotically as PDMS layer thickness is reduced.
    Figure Legend Snippet: Effect of PDMS layer thickness on the flow distance for 1.0 μg/mL streptavidin in biotin-modified 17 μm deep and 58 μm wide channels. Flow distance decreases asymptotically as PDMS layer thickness is reduced.

    Techniques Used: Flow Cytometry, Modification

    Plots of background subtracted, normalized fluorescence signal and peak area in 13 μm deep biotin-modified channels as a function of channel position and flow distance to probe constriction. (A–B) Unlabeled streptavidin (500 μg/mL)
    Figure Legend Snippet: Plots of background subtracted, normalized fluorescence signal and peak area in 13 μm deep biotin-modified channels as a function of channel position and flow distance to probe constriction. (A–B) Unlabeled streptavidin (500 μg/mL)

    Techniques Used: Fluorescence, Modification, Flow Cytometry

    37) Product Images from "Release of human TFIIB from actively transcribing complexes is triggered upon synthesis of 7 nt and 9 nt RNAs"

    Article Title: Release of human TFIIB from actively transcribing complexes is triggered upon synthesis of 7 nt and 9 nt RNAs

    Journal: bioRxiv

    doi: 10.1101/2019.12.19.882902

    TFIIB releases from nearly all transcriptionally active complexes. A. Schematic of the single molecule transcription assay. PICs are immobilized on slides through a biotin on the long non-template strand oligo that binds a streptavidin-derivatized slide surface. The quencher oligo is displaced by transcribing Pol II, resulting in the appearance of Cy3 fluorescence. The fate of AF647-TFIIB in the active complexes can be determined from emission in the red channel. B. Single molecule co-localization of AF647-TFIIB emission and Cy3-DNA emission is highly specific. Plotted are AF647 (red) and Cy3 (green) co-localized spots in PICs and after one image was rotated 90°. Each bar is the average of 4 regions and the errors are the standard deviations. C. Single molecule intensity traces of Cy3 and AF647 emission from co-localized green/red spot pairs were used to determine the fate of AF647-TFIIB in active transcription complexes after the addition of NTPs. Shown are representative traces from active complexes that either released TFIIB (top) or retained TFIIB (bottom). To create the plots, 25 frames of PIC emission were merged with 25 frames of emission after NTPs. D. The majority of transcriptionally active complexes release TFIIB after addition of NTPs. Data are from 248 co-localized spot pairs representing active complexes, plotted is the average of 4 regions and the errors are the standard deviations.
    Figure Legend Snippet: TFIIB releases from nearly all transcriptionally active complexes. A. Schematic of the single molecule transcription assay. PICs are immobilized on slides through a biotin on the long non-template strand oligo that binds a streptavidin-derivatized slide surface. The quencher oligo is displaced by transcribing Pol II, resulting in the appearance of Cy3 fluorescence. The fate of AF647-TFIIB in the active complexes can be determined from emission in the red channel. B. Single molecule co-localization of AF647-TFIIB emission and Cy3-DNA emission is highly specific. Plotted are AF647 (red) and Cy3 (green) co-localized spots in PICs and after one image was rotated 90°. Each bar is the average of 4 regions and the errors are the standard deviations. C. Single molecule intensity traces of Cy3 and AF647 emission from co-localized green/red spot pairs were used to determine the fate of AF647-TFIIB in active transcription complexes after the addition of NTPs. Shown are representative traces from active complexes that either released TFIIB (top) or retained TFIIB (bottom). To create the plots, 25 frames of PIC emission were merged with 25 frames of emission after NTPs. D. The majority of transcriptionally active complexes release TFIIB after addition of NTPs. Data are from 248 co-localized spot pairs representing active complexes, plotted is the average of 4 regions and the errors are the standard deviations.

    Techniques Used: Fluorescence

    38) Product Images from "Selection of Single-Stranded DNA Molecular Recognition Elements against Exotoxin A Using a Novel Decoy-SELEX Method and Sensitive Detection of Exotoxin A in Human Serum"

    Article Title: Selection of Single-Stranded DNA Molecular Recognition Elements against Exotoxin A Using a Novel Decoy-SELEX Method and Sensitive Detection of Exotoxin A in Human Serum

    Journal: BioMed Research International

    doi: 10.1155/2015/417641

    Structures of targets used in the Decoy-SELEX and SPR cross-binding assays. (a) Ribbon structure of the target of interest, Exotoxin A (PDB 1IKQ, 66 kDa) [ 8 ]. (b) Ribbon structure of streptavidin (PDB 4GJS, 60 kDa) used in cross bind assays [ 23 ]. ((c), (d)) Ribbon structures of bovine serum albumin (PDB 4F5S, 66.5 kDa) and Cholera toxin (PDB 2A5D, 84 kDa) used in negative rounds of selection and crossing binding assays [ 24 , 25 ]. (e) Chemical structure of biotin used in negative rounds of selection and cross-binding assays.
    Figure Legend Snippet: Structures of targets used in the Decoy-SELEX and SPR cross-binding assays. (a) Ribbon structure of the target of interest, Exotoxin A (PDB 1IKQ, 66 kDa) [ 8 ]. (b) Ribbon structure of streptavidin (PDB 4GJS, 60 kDa) used in cross bind assays [ 23 ]. ((c), (d)) Ribbon structures of bovine serum albumin (PDB 4F5S, 66.5 kDa) and Cholera toxin (PDB 2A5D, 84 kDa) used in negative rounds of selection and crossing binding assays [ 24 , 25 ]. (e) Chemical structure of biotin used in negative rounds of selection and cross-binding assays.

    Techniques Used: SPR Assay, Binding Assay, Selection

    39) Product Images from "The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans"

    Article Title: The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2001164

    Mlh1-Mlh3’s endonuclease activity requires a continuous substrate and increases as substrate size increases. (A) Denaturing agarose analysis of yeast Mlh1-Mlh3 nicking on circular pUC18 (linearized prior to gel loading) (2.7 kb; black), Hin dIII linearized pUC18 (red), and Hin dIII linearized pUC18 with streptavidin (SA) bound to ends (blue). Migration of linearized substrate (l) is indicated. (B) Average of two separate experiments: fraction nicked defined as fraction of substrate lost plotted against yeast Mlh1-Mlh3 concentration; error bars represent the standard deviation between three experiments. (C) Top: native agarose gel electrophoresis analysis of yeast Mlh1-Mlh3 (150 nM) endonuclease activity on circular substrate ranging from 2.7 kb to 12 kb. The concentration of nucleotide in each reaction is 15 μM. (D) Quantification of nicking in lanes 4, 7, 10, and 13 in C averaged from three separate experiments. Error bars indicate standard deviation. (E) Denaturing agarose analysis of yeast Mlh1-Mlh3 nicking on 12-kb circular DNA (black) and Hin dIII linearized 12 kb substrate (red). (F) Average of three separate experiments; error bars represent standard deviation. All nicking reactions were carried out for 60 min.
    Figure Legend Snippet: Mlh1-Mlh3’s endonuclease activity requires a continuous substrate and increases as substrate size increases. (A) Denaturing agarose analysis of yeast Mlh1-Mlh3 nicking on circular pUC18 (linearized prior to gel loading) (2.7 kb; black), Hin dIII linearized pUC18 (red), and Hin dIII linearized pUC18 with streptavidin (SA) bound to ends (blue). Migration of linearized substrate (l) is indicated. (B) Average of two separate experiments: fraction nicked defined as fraction of substrate lost plotted against yeast Mlh1-Mlh3 concentration; error bars represent the standard deviation between three experiments. (C) Top: native agarose gel electrophoresis analysis of yeast Mlh1-Mlh3 (150 nM) endonuclease activity on circular substrate ranging from 2.7 kb to 12 kb. The concentration of nucleotide in each reaction is 15 μM. (D) Quantification of nicking in lanes 4, 7, 10, and 13 in C averaged from three separate experiments. Error bars indicate standard deviation. (E) Denaturing agarose analysis of yeast Mlh1-Mlh3 nicking on 12-kb circular DNA (black) and Hin dIII linearized 12 kb substrate (red). (F) Average of three separate experiments; error bars represent standard deviation. All nicking reactions were carried out for 60 min.

    Techniques Used: Activity Assay, Migration, Concentration Assay, Standard Deviation, Agarose Gel Electrophoresis

    Mlh1-Mlh3’s endonuclease activity is inhibited by a loop mismatch and biotin-streptavidin linkages in plasmid DNA. (A) Mlh1-Mlh3 nicking activity on homoduplex (black), biotinylated (green), or biotin-streptavidin–containing (blue) 7.2 kb circular substrates (15 μM total nucleotide). Lanes 4–7, 10–13, and 16–19 contain 50, 150, 300, and 500 nM Mlh1-Mlh3, respectively. The amount of nicked product (n) and linear product (black triangle) was quantified as a fraction of the total starting closed circular substrate (cc). (B) Average of three separate experiments is plotted. Error bars indicate the standard deviation. (C) Mlh1-Mlh3 nicking activity on homoduplex (black) or +8 loop mismatch–containing (red) 7 kb circular substrate (15 μM total nucleotide). Lanes 3–7 and 9–13 contain 25, 50, 150, 200, and 300 nM Mlh1-Mlh3, respectively. (D) Average of three separate experiments is plotted. Error bars indicate the standard deviation. (E) Mlh1-Mlh3 nicking activity on 7 kb linear substrates containing a +8 loop mismatch 550 ( Drd I), 3,900 ( Afe I), or 6,600 ( Bgl II) base pairs from one end. Average of two experiments is indicated below the gel. See Materials and methods for details. All nicking reactions were carried out for 60 min.
    Figure Legend Snippet: Mlh1-Mlh3’s endonuclease activity is inhibited by a loop mismatch and biotin-streptavidin linkages in plasmid DNA. (A) Mlh1-Mlh3 nicking activity on homoduplex (black), biotinylated (green), or biotin-streptavidin–containing (blue) 7.2 kb circular substrates (15 μM total nucleotide). Lanes 4–7, 10–13, and 16–19 contain 50, 150, 300, and 500 nM Mlh1-Mlh3, respectively. The amount of nicked product (n) and linear product (black triangle) was quantified as a fraction of the total starting closed circular substrate (cc). (B) Average of three separate experiments is plotted. Error bars indicate the standard deviation. (C) Mlh1-Mlh3 nicking activity on homoduplex (black) or +8 loop mismatch–containing (red) 7 kb circular substrate (15 μM total nucleotide). Lanes 3–7 and 9–13 contain 25, 50, 150, 200, and 300 nM Mlh1-Mlh3, respectively. (D) Average of three separate experiments is plotted. Error bars indicate the standard deviation. (E) Mlh1-Mlh3 nicking activity on 7 kb linear substrates containing a +8 loop mismatch 550 ( Drd I), 3,900 ( Afe I), or 6,600 ( Bgl II) base pairs from one end. Average of two experiments is indicated below the gel. See Materials and methods for details. All nicking reactions were carried out for 60 min.

    Techniques Used: Activity Assay, Plasmid Preparation, Standard Deviation

    40) Product Images from "IR-783 Labeling of a Peptide Receptor for ‘Turn-On’ Fluorescence Based Sensing"

    Article Title: IR-783 Labeling of a Peptide Receptor for ‘Turn-On’ Fluorescence Based Sensing

    Journal: Chemosensors (Basel, Switzerland)

    doi: 10.3390/chemosensors6040047

    Schematic of IR-783 labeled peptide sequence VSHPQAPF serving as biotin mimic for recognition by streptavidin. Binding results in induction of turn-on fluorescence enhancement that is reversible upon addition of native biotin to displace the probe returning it to weak fluorescence.
    Figure Legend Snippet: Schematic of IR-783 labeled peptide sequence VSHPQAPF serving as biotin mimic for recognition by streptavidin. Binding results in induction of turn-on fluorescence enhancement that is reversible upon addition of native biotin to displace the probe returning it to weak fluorescence.

    Techniques Used: Labeling, Sequencing, Binding Assay, Fluorescence

    The fluorescence intensity of the IR783-VSHPQAPF was observed to increase linearly with increasing concentrations of streptavidin.
    Figure Legend Snippet: The fluorescence intensity of the IR783-VSHPQAPF was observed to increase linearly with increasing concentrations of streptavidin.

    Techniques Used: Fluorescence

    The biotin mimetic probe IR-783 VSHPQAPF, when bound to streptavidin, has enhanced fluorescence, which is reversible when native biotin is subsequently added to competitively elute the probe from the streptavidin target: ( a ) Addition of increasing amount of biotin shows a significant decrease in fluorescence when more than one equivalent of biotin was combined with respect to the streptavidin concentration; ( b ) Fluorescence spectra show that the addition of 3 equivalents of biotin results in a decrease in intensity of the signal but no significant peak shift.
    Figure Legend Snippet: The biotin mimetic probe IR-783 VSHPQAPF, when bound to streptavidin, has enhanced fluorescence, which is reversible when native biotin is subsequently added to competitively elute the probe from the streptavidin target: ( a ) Addition of increasing amount of biotin shows a significant decrease in fluorescence when more than one equivalent of biotin was combined with respect to the streptavidin concentration; ( b ) Fluorescence spectra show that the addition of 3 equivalents of biotin results in a decrease in intensity of the signal but no significant peak shift.

    Techniques Used: Fluorescence, Concentration Assay

    Microscale thermophoresis signals resulting from binding induced changes in hydration shell corresponding to distinct thermophoretic signals for the probe (IR783-VSHPQAPF) with increasing concentrations of streptavidin target.
    Figure Legend Snippet: Microscale thermophoresis signals resulting from binding induced changes in hydration shell corresponding to distinct thermophoretic signals for the probe (IR783-VSHPQAPF) with increasing concentrations of streptavidin target.

    Techniques Used: Microscale Thermophoresis, Binding Assay

    Microscale thermophoresis showing distinct signals for the IR783-VSHPQAPF probe before and after binding to streptavidin revealing a clear binding event and again returning to its original signal upon addition of native biotin confirming the release of the probe from streptavidin.
    Figure Legend Snippet: Microscale thermophoresis showing distinct signals for the IR783-VSHPQAPF probe before and after binding to streptavidin revealing a clear binding event and again returning to its original signal upon addition of native biotin confirming the release of the probe from streptavidin.

    Techniques Used: Microscale Thermophoresis, Binding Assay

    Related Articles

    Produced:

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    Transfection:

    Article Title: La-related protein 1 (LARP1) repression of TOP mRNA translation is mediated through its cap-binding domain and controlled by an adjacent regulatory region
    Article Snippet: .. Materials Reagents were obtained from the following sources: antibodies for S6K, phospho-T389-S6K, eIF2α, phospho-Ser51-eIF2α, Raptor, mTOR, 4EBP1, LARP1, NCBP1, eIF4E, eIF4G and PABP from Cell Signaling Technology; primary antibodies for eIF3b and HRP-labeled secondary antibodies from Santa Cruz Biotechnology; IRDye secondary antibodies from LI-COR; Dulbecco’s modified Eagle’s medium (DMEM) from Life Technologies; heat-inactivated Fetal Bovine Serum (IFS) and 7mGDP from Sigma Aldrich; DNase I, T4 DNA ligase 1, T4 RNA Ligase I, T7 RNA polymerase, polynucleotide kinase, Protoscript II reverse transcriptase, Vaccinia Capping System, Oligo d(T)25 Magnetic beads and streptavidin-coated magnetic beads from New England Biolabs; iTaq Universal SYBR Green Supermix and Bradford Protein Assay from Bio-rad; RNeasy Plus Mini Kit from Qiagen; Dual Luciferase Assay from Promega; and X-tremeGENE 9 transfection reagent from Roche. .. Cell culture and preparation of cell extracts Cells were grown in high-glucose DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum and penicillin-streptomycin.

    Luciferase:

    Article Title: La-related protein 1 (LARP1) repression of TOP mRNA translation is mediated through its cap-binding domain and controlled by an adjacent regulatory region
    Article Snippet: .. Materials Reagents were obtained from the following sources: antibodies for S6K, phospho-T389-S6K, eIF2α, phospho-Ser51-eIF2α, Raptor, mTOR, 4EBP1, LARP1, NCBP1, eIF4E, eIF4G and PABP from Cell Signaling Technology; primary antibodies for eIF3b and HRP-labeled secondary antibodies from Santa Cruz Biotechnology; IRDye secondary antibodies from LI-COR; Dulbecco’s modified Eagle’s medium (DMEM) from Life Technologies; heat-inactivated Fetal Bovine Serum (IFS) and 7mGDP from Sigma Aldrich; DNase I, T4 DNA ligase 1, T4 RNA Ligase I, T7 RNA polymerase, polynucleotide kinase, Protoscript II reverse transcriptase, Vaccinia Capping System, Oligo d(T)25 Magnetic beads and streptavidin-coated magnetic beads from New England Biolabs; iTaq Universal SYBR Green Supermix and Bradford Protein Assay from Bio-rad; RNeasy Plus Mini Kit from Qiagen; Dual Luciferase Assay from Promega; and X-tremeGENE 9 transfection reagent from Roche. .. Cell culture and preparation of cell extracts Cells were grown in high-glucose DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum and penicillin-streptomycin.

    Synthesized:

    Article Title: A Novel mRNA Level Subtraction Method for Quick Identification of Target-Orientated Uniquely Expressed Genes Between Peanut Immature Pod and Leaf
    Article Snippet: .. Streptavidin-coated magnetic beads (1.1 mg/ml) were applied again to purify synthesized double-stranded cDNAs with three times washes. .. The double-stranded cDNAs were released from the magnetic beads by Not I (75 U) restriction digestion at 37°C for 1 h followed by the same volume phenol/chloroform/isoamyl alcohol (25:24:1) extraction and precipitation using 7.5 M sodium acetate, glycogen (20 μg/μl), and ice-cold absolute ethanol at -20°C overnight.

    Magnetic Beads:

    Article Title: La-related protein 1 (LARP1) repression of TOP mRNA translation is mediated through its cap-binding domain and controlled by an adjacent regulatory region
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    Article Title: A Novel mRNA Level Subtraction Method for Quick Identification of Target-Orientated Uniquely Expressed Genes Between Peanut Immature Pod and Leaf
    Article Snippet: .. Streptavidin-coated magnetic beads (1.1 mg/ml) were applied again to purify synthesized double-stranded cDNAs with three times washes. .. The double-stranded cDNAs were released from the magnetic beads by Not I (75 U) restriction digestion at 37°C for 1 h followed by the same volume phenol/chloroform/isoamyl alcohol (25:24:1) extraction and precipitation using 7.5 M sodium acetate, glycogen (20 μg/μl), and ice-cold absolute ethanol at -20°C overnight.

    Article Title: Simultaneous and stoichiometric purification of hundreds of oligonucleotides
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    SYBR Green Assay:

    Article Title: La-related protein 1 (LARP1) repression of TOP mRNA translation is mediated through its cap-binding domain and controlled by an adjacent regulatory region
    Article Snippet: .. Materials Reagents were obtained from the following sources: antibodies for S6K, phospho-T389-S6K, eIF2α, phospho-Ser51-eIF2α, Raptor, mTOR, 4EBP1, LARP1, NCBP1, eIF4E, eIF4G and PABP from Cell Signaling Technology; primary antibodies for eIF3b and HRP-labeled secondary antibodies from Santa Cruz Biotechnology; IRDye secondary antibodies from LI-COR; Dulbecco’s modified Eagle’s medium (DMEM) from Life Technologies; heat-inactivated Fetal Bovine Serum (IFS) and 7mGDP from Sigma Aldrich; DNase I, T4 DNA ligase 1, T4 RNA Ligase I, T7 RNA polymerase, polynucleotide kinase, Protoscript II reverse transcriptase, Vaccinia Capping System, Oligo d(T)25 Magnetic beads and streptavidin-coated magnetic beads from New England Biolabs; iTaq Universal SYBR Green Supermix and Bradford Protein Assay from Bio-rad; RNeasy Plus Mini Kit from Qiagen; Dual Luciferase Assay from Promega; and X-tremeGENE 9 transfection reagent from Roche. .. Cell culture and preparation of cell extracts Cells were grown in high-glucose DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum and penicillin-streptomycin.

    Concentration Assay:

    Article Title: Proteins mediating DNA loops effectively block transcription
    Article Snippet: .. Complexes of RNA polymerase bound at the promoter (PC) were produced by incubating 1 nM of DNA with 0.1 μM streptavidin, 7.5 nM LacI (unless otherwise stated) and RNA polymerase holoenzyme (New England Biolabs, Ipswitch, MA) diluted 200 times in transcription buffer (TXB; 20 mM Tris‐glutamate (pH 8.0), 10 mM magnesium‐glutamate, 50 mM potassium‐glutamate, 1mM DTT) for 30 min at 37 ° C. To initiate transcription, the reaction mixture was spiked with 1 mM NTPs to give a final concentration of 100 μM, and incubating at 37 ° C for 60 s. Elongation was terminated by spiking the mixture with 250 mM EDTA in TXB to give a final concentration of 20 mM EDTA, and incubating at 37 ° C for 30 s. 5 μl of the sample solution containing DNA and proteins were deposited on the poly‐L‐ornithine‐coated mica and incubated for 2 min. .. This droplet was rinsed with 400 μl of high‐performance liquid chromatography grade water and dried gently with compressed air.

    Incubation:

    Article Title: Proteins mediating DNA loops effectively block transcription
    Article Snippet: .. Complexes of RNA polymerase bound at the promoter (PC) were produced by incubating 1 nM of DNA with 0.1 μM streptavidin, 7.5 nM LacI (unless otherwise stated) and RNA polymerase holoenzyme (New England Biolabs, Ipswitch, MA) diluted 200 times in transcription buffer (TXB; 20 mM Tris‐glutamate (pH 8.0), 10 mM magnesium‐glutamate, 50 mM potassium‐glutamate, 1mM DTT) for 30 min at 37 ° C. To initiate transcription, the reaction mixture was spiked with 1 mM NTPs to give a final concentration of 100 μM, and incubating at 37 ° C for 60 s. Elongation was terminated by spiking the mixture with 250 mM EDTA in TXB to give a final concentration of 20 mM EDTA, and incubating at 37 ° C for 30 s. 5 μl of the sample solution containing DNA and proteins were deposited on the poly‐L‐ornithine‐coated mica and incubated for 2 min. .. This droplet was rinsed with 400 μl of high‐performance liquid chromatography grade water and dried gently with compressed air.

    other:

    Article Title: "Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation
    Article Snippet: In a revised device design with slightly taller microchannels (5.2 μm) and a PDMS cover layer thickness of 0.7 mm, a 1.0 mg/mL streptavidin solution traveled 10 mm, and a 0.88 mg/mL streptavidin solution traveled 15 mm, while solutions lacking streptavidin flowed the full length (35 mm) of the b-BSA coated channel.

    Bradford Protein Assay:

    Article Title: La-related protein 1 (LARP1) repression of TOP mRNA translation is mediated through its cap-binding domain and controlled by an adjacent regulatory region
    Article Snippet: .. Materials Reagents were obtained from the following sources: antibodies for S6K, phospho-T389-S6K, eIF2α, phospho-Ser51-eIF2α, Raptor, mTOR, 4EBP1, LARP1, NCBP1, eIF4E, eIF4G and PABP from Cell Signaling Technology; primary antibodies for eIF3b and HRP-labeled secondary antibodies from Santa Cruz Biotechnology; IRDye secondary antibodies from LI-COR; Dulbecco’s modified Eagle’s medium (DMEM) from Life Technologies; heat-inactivated Fetal Bovine Serum (IFS) and 7mGDP from Sigma Aldrich; DNase I, T4 DNA ligase 1, T4 RNA Ligase I, T7 RNA polymerase, polynucleotide kinase, Protoscript II reverse transcriptase, Vaccinia Capping System, Oligo d(T)25 Magnetic beads and streptavidin-coated magnetic beads from New England Biolabs; iTaq Universal SYBR Green Supermix and Bradford Protein Assay from Bio-rad; RNeasy Plus Mini Kit from Qiagen; Dual Luciferase Assay from Promega; and X-tremeGENE 9 transfection reagent from Roche. .. Cell culture and preparation of cell extracts Cells were grown in high-glucose DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum and penicillin-streptomycin.

    Modification:

    Article Title: La-related protein 1 (LARP1) repression of TOP mRNA translation is mediated through its cap-binding domain and controlled by an adjacent regulatory region
    Article Snippet: .. Materials Reagents were obtained from the following sources: antibodies for S6K, phospho-T389-S6K, eIF2α, phospho-Ser51-eIF2α, Raptor, mTOR, 4EBP1, LARP1, NCBP1, eIF4E, eIF4G and PABP from Cell Signaling Technology; primary antibodies for eIF3b and HRP-labeled secondary antibodies from Santa Cruz Biotechnology; IRDye secondary antibodies from LI-COR; Dulbecco’s modified Eagle’s medium (DMEM) from Life Technologies; heat-inactivated Fetal Bovine Serum (IFS) and 7mGDP from Sigma Aldrich; DNase I, T4 DNA ligase 1, T4 RNA Ligase I, T7 RNA polymerase, polynucleotide kinase, Protoscript II reverse transcriptase, Vaccinia Capping System, Oligo d(T)25 Magnetic beads and streptavidin-coated magnetic beads from New England Biolabs; iTaq Universal SYBR Green Supermix and Bradford Protein Assay from Bio-rad; RNeasy Plus Mini Kit from Qiagen; Dual Luciferase Assay from Promega; and X-tremeGENE 9 transfection reagent from Roche. .. Cell culture and preparation of cell extracts Cells were grown in high-glucose DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum and penicillin-streptomycin.

    Western Blot:

    Article Title: An efficient and sensitive method for preparing cDNA libraries from scarce biological samples
    Article Snippet: .. 5.0 μl magnetic hydrophilic streptavidin beads (New England Biolabs) were washed three times with 50 μl buffer WB (0.5M NaCl, 20 mM Tris-HCl pH 7.5, 1.0 mM EDTA), resuspended in 5.0 μl TE + 0.3M NaCl and added to each sample. .. After overnight elution, the bead-containing supernatant was transferred to a clean, low-retention 1.7 ml eppendorf tube, tubes were magnetized on a magnetic rack (Life Technologies), supernatants carefully removed, beads washed three times with 1.0 ml buffer WB (magnetizing and carefully removing supernatant between each wash step) and resuspended in 10.0 μl RNase free H2O.

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    New England Biolabs streptavidin coated magnetic beads
    General scheme applied for identifying peanut immature pod-specific genes (tracer mRNA (1)) after a single round subtraction . B biotin, S <t>streptavidin,</t> M magnetic bead.
    Streptavidin Coated Magnetic Beads, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    General scheme applied for identifying peanut immature pod-specific genes (tracer mRNA (1)) after a single round subtraction . B biotin, S streptavidin, M magnetic bead.

    Journal: Biological Procedures Online

    Article Title: A Novel mRNA Level Subtraction Method for Quick Identification of Target-Orientated Uniquely Expressed Genes Between Peanut Immature Pod and Leaf

    doi: 10.1007/s12575-009-9022-z

    Figure Lengend Snippet: General scheme applied for identifying peanut immature pod-specific genes (tracer mRNA (1)) after a single round subtraction . B biotin, S streptavidin, M magnetic bead.

    Article Snippet: Streptavidin-coated magnetic beads (1.1 mg/ml) were applied again to purify synthesized double-stranded cDNAs with three times washes.

    Techniques:

    Immobilization of scFv5- linker-BCCP constructs and scFv5-linker-AviTag constructs onto streptavidin-coated plates. Serial dilutions of cell lysates were loaded onto the coated 96-well plates, and the immobilization was detected with an anti-FLAG antibody. Data are normalized to the signal from the highest concentration of scFv5-BCCP with no linker for each biological replicate (n = 6). The error bars represent the standard error of the mean.

    Journal: Biotechnology and applied biochemistry

    Article Title: Effect of linkers on immobilization of scFvs with biotin-streptavidin interaction

    doi: 10.1002/bab.1645

    Figure Lengend Snippet: Immobilization of scFv5- linker-BCCP constructs and scFv5-linker-AviTag constructs onto streptavidin-coated plates. Serial dilutions of cell lysates were loaded onto the coated 96-well plates, and the immobilization was detected with an anti-FLAG antibody. Data are normalized to the signal from the highest concentration of scFv5-BCCP with no linker for each biological replicate (n = 6). The error bars represent the standard error of the mean.

    Article Snippet: High-binding 96-well polystyrene plates (Corning) were coated with streptavidin (NEB), as previously described [ ].

    Techniques: Construct, Concentration Assay

    Immobilization of scFv13R4-linker-BCCP constructs and scFv13R4-linker-AviTag constructs onto streptavidin-coated plates. Serial dilutions of cell lysates were loaded onto the coated 96-well plates, and the immobilization was detected with an anti-FLAG antibody. Data are normalized to the signal from the highest concentration of scFv13R4-BCCP with no linker for each biological replicate (n = 5). The error bars represent the standard error of the mean.

    Journal: Biotechnology and applied biochemistry

    Article Title: Effect of linkers on immobilization of scFvs with biotin-streptavidin interaction

    doi: 10.1002/bab.1645

    Figure Lengend Snippet: Immobilization of scFv13R4-linker-BCCP constructs and scFv13R4-linker-AviTag constructs onto streptavidin-coated plates. Serial dilutions of cell lysates were loaded onto the coated 96-well plates, and the immobilization was detected with an anti-FLAG antibody. Data are normalized to the signal from the highest concentration of scFv13R4-BCCP with no linker for each biological replicate (n = 5). The error bars represent the standard error of the mean.

    Article Snippet: High-binding 96-well polystyrene plates (Corning) were coated with streptavidin (NEB), as previously described [ ].

    Techniques: Construct, Concentration Assay

    Overview of 3-day generation of cDNA libraries. ( A ) On the first day, total RNA is ligated to a 3′ adapter and cDNA is generated by reverse transcription by tandem reactions in a single tube, RNA is degraded and cDNAs are isolated by ethanol precipitation. ( B ) On the second day, cDNAs are circularized, size selected by gel fractionation and eluted overnight in the presence of streptavidin beads. ( C ) PCR is done on bead-bound purified cDNAs to generate templates ready for high-throughput sequencing.

    Journal: Nucleic Acids Research

    Article Title: An efficient and sensitive method for preparing cDNA libraries from scarce biological samples

    doi: 10.1093/nar/gku637

    Figure Lengend Snippet: Overview of 3-day generation of cDNA libraries. ( A ) On the first day, total RNA is ligated to a 3′ adapter and cDNA is generated by reverse transcription by tandem reactions in a single tube, RNA is degraded and cDNAs are isolated by ethanol precipitation. ( B ) On the second day, cDNAs are circularized, size selected by gel fractionation and eluted overnight in the presence of streptavidin beads. ( C ) PCR is done on bead-bound purified cDNAs to generate templates ready for high-throughput sequencing.

    Article Snippet: 5.0 μl magnetic hydrophilic streptavidin beads (New England Biolabs) were washed three times with 50 μl buffer WB (0.5M NaCl, 20 mM Tris-HCl pH 7.5, 1.0 mM EDTA), resuspended in 5.0 μl TE + 0.3M NaCl and added to each sample.

    Techniques: Generated, Isolation, Ethanol Precipitation, Fractionation, Polymerase Chain Reaction, Purification, Next-Generation Sequencing

    Detailed LQ cloning method. ( A ) A pre-adenylated (rApp) 3′-terminal dideoxy-C (ddC) blocked adapter (gray) is annealed to a ssDNA reverse transcription (RT) oligo (black) in a 1:1 molar ratio. The annealed adapter is ligated to 3′-hydroxyl-containing RNA (orange) using T4 RNA Ligase 2 (truncated K227Q) without ATP. Each RT oligo contains a 5′ Guanine (G) followed by a 4 or 6 nucleotide randomer (N X ), a 3–6 nucleotide barcode (BAR) and 3 internal deoxyUridine (dU) nucleotides. The adapter::RT oligo hybrid is in excess over RNA, resulting in free adapter::primer material present in the completed reaction. ( B ) Reverse transcription of ligated RNA is carried out in the same tube as the ligation reaction generating ‘+ insert’ and ‘no insert’ cDNA products (red and black line) using dGTP, dTTP, dATP, dCTP as well as biotinylated dATP and dCTP (yellow ‘B’-containing circles). The RNA template is degraded (dashed orange line) by base hydrolysis and cDNA is ethanol precipitated with ammonium acetate to facilitate maximum removal of free adapter and unincorporated nucleotides ( C ). Ethanol precipitated cDNAs are circularized ( D ) and resolved on a 10% denaturing polyacrylamide gel. ‘+ insert’ circularized cDNAs are isolated by excising and eluting them from the gel overnight in the presence of magnetic streptavidin beads ( E ). Bead-bound ‘+ insert’ cDNAs serve as templates in the first round of PCR. Amplification is done using a mix containing uracil-N-deglycosylase (UNG) to remove dU nucleotides, thereby generating a linear template through strand scission, and with primers complimentary to the 3′ adapter (blue) and 5′ end of the RT oligo (tan) ( F ). First round PCR products are resolved on an 8% native polyacrylamide gel, the 60–70 nucleotide products are excised and a portion is used as the template for second round PCR. Second round PCR products are generated using primers complimentary to the 3′ adapter (dark blue) and 5′ end of the RT oligo (brown) that contain the full Illumina or Ion Torrent adapter sequences (dark blue and brown) ( G ).

    Journal: Nucleic Acids Research

    Article Title: An efficient and sensitive method for preparing cDNA libraries from scarce biological samples

    doi: 10.1093/nar/gku637

    Figure Lengend Snippet: Detailed LQ cloning method. ( A ) A pre-adenylated (rApp) 3′-terminal dideoxy-C (ddC) blocked adapter (gray) is annealed to a ssDNA reverse transcription (RT) oligo (black) in a 1:1 molar ratio. The annealed adapter is ligated to 3′-hydroxyl-containing RNA (orange) using T4 RNA Ligase 2 (truncated K227Q) without ATP. Each RT oligo contains a 5′ Guanine (G) followed by a 4 or 6 nucleotide randomer (N X ), a 3–6 nucleotide barcode (BAR) and 3 internal deoxyUridine (dU) nucleotides. The adapter::RT oligo hybrid is in excess over RNA, resulting in free adapter::primer material present in the completed reaction. ( B ) Reverse transcription of ligated RNA is carried out in the same tube as the ligation reaction generating ‘+ insert’ and ‘no insert’ cDNA products (red and black line) using dGTP, dTTP, dATP, dCTP as well as biotinylated dATP and dCTP (yellow ‘B’-containing circles). The RNA template is degraded (dashed orange line) by base hydrolysis and cDNA is ethanol precipitated with ammonium acetate to facilitate maximum removal of free adapter and unincorporated nucleotides ( C ). Ethanol precipitated cDNAs are circularized ( D ) and resolved on a 10% denaturing polyacrylamide gel. ‘+ insert’ circularized cDNAs are isolated by excising and eluting them from the gel overnight in the presence of magnetic streptavidin beads ( E ). Bead-bound ‘+ insert’ cDNAs serve as templates in the first round of PCR. Amplification is done using a mix containing uracil-N-deglycosylase (UNG) to remove dU nucleotides, thereby generating a linear template through strand scission, and with primers complimentary to the 3′ adapter (blue) and 5′ end of the RT oligo (tan) ( F ). First round PCR products are resolved on an 8% native polyacrylamide gel, the 60–70 nucleotide products are excised and a portion is used as the template for second round PCR. Second round PCR products are generated using primers complimentary to the 3′ adapter (dark blue) and 5′ end of the RT oligo (brown) that contain the full Illumina or Ion Torrent adapter sequences (dark blue and brown) ( G ).

    Article Snippet: 5.0 μl magnetic hydrophilic streptavidin beads (New England Biolabs) were washed three times with 50 μl buffer WB (0.5M NaCl, 20 mM Tris-HCl pH 7.5, 1.0 mM EDTA), resuspended in 5.0 μl TE + 0.3M NaCl and added to each sample.

    Techniques: Clone Assay, Ligation, Isolation, Polymerase Chain Reaction, Amplification, Generated

    Flow distance traveled as a function of streptavidin concentration in biotin-modified microchannels (58 μm wide) of two different heights, with a 0.5 mm thick PDMS top layer. (A) 13 μm deep channels. (B) 17 μm deep channels.

    Journal: Analytical chemistry

    Article Title: "Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation

    doi: 10.1021/ac301278s

    Figure Lengend Snippet: Flow distance traveled as a function of streptavidin concentration in biotin-modified microchannels (58 μm wide) of two different heights, with a 0.5 mm thick PDMS top layer. (A) 13 μm deep channels. (B) 17 μm deep channels.

    Article Snippet: In a revised device design with slightly taller microchannels (5.2 μm) and a PDMS cover layer thickness of 0.7 mm, a 1.0 mg/mL streptavidin solution traveled 10 mm, and a 0.88 mg/mL streptavidin solution traveled 15 mm, while solutions lacking streptavidin flowed the full length (35 mm) of the b-BSA coated channel.

    Techniques: Flow Cytometry, Concentration Assay, Modification

    Effect of solution viscosity on the flow distance of 100 ng/mL (X) and 1.0 μg/mL (O) streptavidin solutions in biotin-modified 17 μm deep and 58 μm wide channels with a 0.5 mm PDMS top layer. Flow distance is only affected above

    Journal: Analytical chemistry

    Article Title: "Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation

    doi: 10.1021/ac301278s

    Figure Lengend Snippet: Effect of solution viscosity on the flow distance of 100 ng/mL (X) and 1.0 μg/mL (O) streptavidin solutions in biotin-modified 17 μm deep and 58 μm wide channels with a 0.5 mm PDMS top layer. Flow distance is only affected above

    Article Snippet: In a revised device design with slightly taller microchannels (5.2 μm) and a PDMS cover layer thickness of 0.7 mm, a 1.0 mg/mL streptavidin solution traveled 10 mm, and a 0.88 mg/mL streptavidin solution traveled 15 mm, while solutions lacking streptavidin flowed the full length (35 mm) of the b-BSA coated channel.

    Techniques: Flow Cytometry, Modification

    Effect of PDMS layer thickness on the flow distance for 1.0 μg/mL streptavidin in biotin-modified 17 μm deep and 58 μm wide channels. Flow distance decreases asymptotically as PDMS layer thickness is reduced.

    Journal: Analytical chemistry

    Article Title: "Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation

    doi: 10.1021/ac301278s

    Figure Lengend Snippet: Effect of PDMS layer thickness on the flow distance for 1.0 μg/mL streptavidin in biotin-modified 17 μm deep and 58 μm wide channels. Flow distance decreases asymptotically as PDMS layer thickness is reduced.

    Article Snippet: In a revised device design with slightly taller microchannels (5.2 μm) and a PDMS cover layer thickness of 0.7 mm, a 1.0 mg/mL streptavidin solution traveled 10 mm, and a 0.88 mg/mL streptavidin solution traveled 15 mm, while solutions lacking streptavidin flowed the full length (35 mm) of the b-BSA coated channel.

    Techniques: Flow Cytometry, Modification

    Plots of background subtracted, normalized fluorescence signal and peak area in 13 μm deep biotin-modified channels as a function of channel position and flow distance to probe constriction. (A–B) Unlabeled streptavidin (500 μg/mL)

    Journal: Analytical chemistry

    Article Title: "Flow valve" microfluidic devices for simple, detectorless and label-free analyte quantitation

    doi: 10.1021/ac301278s

    Figure Lengend Snippet: Plots of background subtracted, normalized fluorescence signal and peak area in 13 μm deep biotin-modified channels as a function of channel position and flow distance to probe constriction. (A–B) Unlabeled streptavidin (500 μg/mL)

    Article Snippet: In a revised device design with slightly taller microchannels (5.2 μm) and a PDMS cover layer thickness of 0.7 mm, a 1.0 mg/mL streptavidin solution traveled 10 mm, and a 0.88 mg/mL streptavidin solution traveled 15 mm, while solutions lacking streptavidin flowed the full length (35 mm) of the b-BSA coated channel.

    Techniques: Fluorescence, Modification, Flow Cytometry