tris  (Thermo Fisher)


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  • 99
    Name:
    Tris
    Description:
    Thermo Scientific Pierce Tris is a standard reagent for preparation of many molecular biology buffers Features of Thermo Scientific Pierce Tris • 99 8 pure • White crystalline powder • Optimal buffering between pH 7 and 9
    Catalog Number:
    17926
    Price:
    None
    Applications:
    DNA & RNA Purification & Analysis
    Category:
    Lab Reagents and Chemicals
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    Structured Review

    Thermo Fisher tris
    HCV helicase catalyzed DNA unwinding in various buffer systems. ( A ) 1 nM of a DNA substrate was incubated with 100 nM of HCV helicase (His–Hel) and 5 mM ATP for 10 min at 23°C in either citrate buffer (squares), MES-NaOH buffer (triangles), PIPES-NaOH buffer (diamonds), <t>MOPS-NaOH</t> buffer (circles), <t>Tris–HCl</t> buffer (×), Tris–Malate buffer (+) or Tricene-HCl ( * ). Data are reported as percent of substrate unwound. ( B ) Native polyacrylamide gels showing the time course of the reactions catalyzed in MOPS buffers at pH 6.25, 7.04 and 7.68. Reactions were terminated at various times are shown along with a boiled substrate control.
    Thermo Scientific Pierce Tris is a standard reagent for preparation of many molecular biology buffers Features of Thermo Scientific Pierce Tris • 99 8 pure • White crystalline powder • Optimal buffering between pH 7 and 9
    https://www.bioz.com/result/tris/product/Thermo Fisher
    Average 99 stars, based on 1287 article reviews
    Price from $9.99 to $1999.99
    tris - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Enhanced nucleic acid binding to ATP-bound hepatitis C virus NS3 helicase at low pH activates RNA unwinding"

    Article Title: Enhanced nucleic acid binding to ATP-bound hepatitis C virus NS3 helicase at low pH activates RNA unwinding

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh743

    HCV helicase catalyzed DNA unwinding in various buffer systems. ( A ) 1 nM of a DNA substrate was incubated with 100 nM of HCV helicase (His–Hel) and 5 mM ATP for 10 min at 23°C in either citrate buffer (squares), MES-NaOH buffer (triangles), PIPES-NaOH buffer (diamonds), MOPS-NaOH buffer (circles), Tris–HCl buffer (×), Tris–Malate buffer (+) or Tricene-HCl ( * ). Data are reported as percent of substrate unwound. ( B ) Native polyacrylamide gels showing the time course of the reactions catalyzed in MOPS buffers at pH 6.25, 7.04 and 7.68. Reactions were terminated at various times are shown along with a boiled substrate control.
    Figure Legend Snippet: HCV helicase catalyzed DNA unwinding in various buffer systems. ( A ) 1 nM of a DNA substrate was incubated with 100 nM of HCV helicase (His–Hel) and 5 mM ATP for 10 min at 23°C in either citrate buffer (squares), MES-NaOH buffer (triangles), PIPES-NaOH buffer (diamonds), MOPS-NaOH buffer (circles), Tris–HCl buffer (×), Tris–Malate buffer (+) or Tricene-HCl ( * ). Data are reported as percent of substrate unwound. ( B ) Native polyacrylamide gels showing the time course of the reactions catalyzed in MOPS buffers at pH 6.25, 7.04 and 7.68. Reactions were terminated at various times are shown along with a boiled substrate control.

    Techniques Used: Incubation

    Effect of pH on HCV helicase catalyzed ATP hydrolysis. ( A ) Turnover rate of ATP hydrolysis catalyzed by His–Hel in the absence of nucleic acid as a function of pH. ( B ) Turnover rate of ATP hydrolysis catalyzed by His–Hel in the presence of saturating 2 mM poly(U) RNA. In both (A) and (B), reactions were performed in MES buffer (squares), PIPES buffer (triangles), MOPS buffer (diamonds), TRIS buffer (circles), Tricene buffer (×) or CAPS buffer (+). Average rate constants from four separate determinations are shown with the standard deviations as error bars.
    Figure Legend Snippet: Effect of pH on HCV helicase catalyzed ATP hydrolysis. ( A ) Turnover rate of ATP hydrolysis catalyzed by His–Hel in the absence of nucleic acid as a function of pH. ( B ) Turnover rate of ATP hydrolysis catalyzed by His–Hel in the presence of saturating 2 mM poly(U) RNA. In both (A) and (B), reactions were performed in MES buffer (squares), PIPES buffer (triangles), MOPS buffer (diamonds), TRIS buffer (circles), Tricene buffer (×) or CAPS buffer (+). Average rate constants from four separate determinations are shown with the standard deviations as error bars.

    Techniques Used:

    2) Product Images from "Thermostable virus portal proteins as reprogrammable adapters for solid-state nanopore sensors"

    Article Title: Thermostable virus portal proteins as reprogrammable adapters for solid-state nanopore sensors

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07116-x

    Characterization of the hybrid pore formation. a Typical current profile over time recorded through a 5.5 nm diameter SS pore at +100 mV. After injection of 0.1 nmol of portal protein, short current drops are detected, interpreted as portal collisions with the solid-state nanopore. b A representative current vs time trace recorded for a 5.4 nm SS nanopore at +80 mV, showing stable insertion of a portal protein. c Current as a function of the applied voltage for a 5.7 nm diameter SS pore recorded before (red markers) and after insertion of a portal protein (purple). d Current noise analysis of a 5.5 nm diameter solid-state nanopore before (red) and after insertion of a portal protein (purple). e Conductance of solid-state nanopore vs conductance of hybrid SS-portal pores formed after electrokinetic corking of the portal protein within the SS nanopore ( n = 32 for CD/N hybrids and n = 15 for CGG hybrids). Experiments were performed in 0.5 M NaCl, 20 mM Tris, pH 7.5
    Figure Legend Snippet: Characterization of the hybrid pore formation. a Typical current profile over time recorded through a 5.5 nm diameter SS pore at +100 mV. After injection of 0.1 nmol of portal protein, short current drops are detected, interpreted as portal collisions with the solid-state nanopore. b A representative current vs time trace recorded for a 5.4 nm SS nanopore at +80 mV, showing stable insertion of a portal protein. c Current as a function of the applied voltage for a 5.7 nm diameter SS pore recorded before (red markers) and after insertion of a portal protein (purple). d Current noise analysis of a 5.5 nm diameter solid-state nanopore before (red) and after insertion of a portal protein (purple). e Conductance of solid-state nanopore vs conductance of hybrid SS-portal pores formed after electrokinetic corking of the portal protein within the SS nanopore ( n = 32 for CD/N hybrids and n = 15 for CGG hybrids). Experiments were performed in 0.5 M NaCl, 20 mM Tris, pH 7.5

    Techniques Used: Injection

    Sensing different biopolymers using a hybrid nanopore. Current vs time trace recorded through the hybrid pore at +60 mV in the presence of a 36.0 μM insulin, b 7.7 μM DNA hairpin, c 10.3 μM TPX2 peptide and d 16.6 μM ssDNA. The data in a were filtered at 10 kHz (gray) or 0.5 kHz (green). e Scatter plot of Δ I vs dwell time for the DNA hairpin (red, n = 5883 events), the peptide (purple, n = 3368 events) and the ssDNA (orange, n = 18,812 events)). Experiments were performed in 0.5 M NaCl, 20 mM Tris, pH 7.5
    Figure Legend Snippet: Sensing different biopolymers using a hybrid nanopore. Current vs time trace recorded through the hybrid pore at +60 mV in the presence of a 36.0 μM insulin, b 7.7 μM DNA hairpin, c 10.3 μM TPX2 peptide and d 16.6 μM ssDNA. The data in a were filtered at 10 kHz (gray) or 0.5 kHz (green). e Scatter plot of Δ I vs dwell time for the DNA hairpin (red, n = 5883 events), the peptide (purple, n = 3368 events) and the ssDNA (orange, n = 18,812 events)). Experiments were performed in 0.5 M NaCl, 20 mM Tris, pH 7.5

    Techniques Used:

    Dynamics of TPX2 peptide transport. a Current vs time trace recorded through a hybrid pore at +30, +40 and +55 mV in the presence of 10.3 μM TPX2 peptide. b Semi-log plot of the event frequency as a function of the applied voltage. c Semi-log plot of the peptide dwell time as a function of the applied voltage. The lines in ( b , c ) are exponential fits to the data. Experiments were performed in 0.5 M NaCl, 20 mM Tris, pH 7.5. Data shown in semi-log plots are mean and s.d. of 23,516 events (total for all points) from one hybrid nanopore
    Figure Legend Snippet: Dynamics of TPX2 peptide transport. a Current vs time trace recorded through a hybrid pore at +30, +40 and +55 mV in the presence of 10.3 μM TPX2 peptide. b Semi-log plot of the event frequency as a function of the applied voltage. c Semi-log plot of the peptide dwell time as a function of the applied voltage. The lines in ( b , c ) are exponential fits to the data. Experiments were performed in 0.5 M NaCl, 20 mM Tris, pH 7.5. Data shown in semi-log plots are mean and s.d. of 23,516 events (total for all points) from one hybrid nanopore

    Techniques Used:

    Design of a bio-inspired lipid-free hybrid nanopore. a Cartoon of the DNA packaging machine of a dsDNA virus. Viral genomic DNA (red) is translocated into the preformed virus capsid by the packaging ATPase (yellow) through the portal protein (aqua) embedded in viral capsid (gray). b Left, electrostatic properties of the tunnel in wild-type and mutant portal proteins. Slice through the middle of molecular surface colored according to charge from red (−1 kT e −1 ) to blue (+1 kT e −1 ). b Right (gray box), dimensions of the portal protein (teal, L) and the SS nanopore (pink, R). c Insertion of the purified portal protein into a nanopore fabricated in a thin solid-state (SS) membrane. Portal protein is applied to the trans chamber of a SS nanopore device containing an electrolyte solution of 20 mM Tris pH 7.5, 0.5 M NaCl. The protein electrokinetically inserts into the SS pore during application of a positive voltage. d Cartoon image of the hybrid pore, in which application of voltage results in ion current through the pore (blue arrows), as well as leakage current that is peripheral to the pore (red arrows)
    Figure Legend Snippet: Design of a bio-inspired lipid-free hybrid nanopore. a Cartoon of the DNA packaging machine of a dsDNA virus. Viral genomic DNA (red) is translocated into the preformed virus capsid by the packaging ATPase (yellow) through the portal protein (aqua) embedded in viral capsid (gray). b Left, electrostatic properties of the tunnel in wild-type and mutant portal proteins. Slice through the middle of molecular surface colored according to charge from red (−1 kT e −1 ) to blue (+1 kT e −1 ). b Right (gray box), dimensions of the portal protein (teal, L) and the SS nanopore (pink, R). c Insertion of the purified portal protein into a nanopore fabricated in a thin solid-state (SS) membrane. Portal protein is applied to the trans chamber of a SS nanopore device containing an electrolyte solution of 20 mM Tris pH 7.5, 0.5 M NaCl. The protein electrokinetically inserts into the SS pore during application of a positive voltage. d Cartoon image of the hybrid pore, in which application of voltage results in ion current through the pore (blue arrows), as well as leakage current that is peripheral to the pore (red arrows)

    Techniques Used: Mutagenesis, Purification

    3) Product Images from "Design and Evaluation of a Multiplexed Assay to Assess Human Immunogenicity Against Humira®"

    Article Title: Design and Evaluation of a Multiplexed Assay to Assess Human Immunogenicity Against Humira®

    Journal: The AAPS Journal

    doi: 10.1208/s12248-020-00487-4

    Enrichment of anti-Humira® reactive antibodies from samples. The figure shows use of biotin-labeled Humira® to capture Humira® reactive ADA from the sample in solution. The ADA biotin-Humira® complexes were then captured on streptavidin-coated plates, washed, and the ADA released into solution using 100 mM acetic acid. The ADA enriched sample was neutralized with 150 mM Tris buffer and analyzed in the multiplexed assay
    Figure Legend Snippet: Enrichment of anti-Humira® reactive antibodies from samples. The figure shows use of biotin-labeled Humira® to capture Humira® reactive ADA from the sample in solution. The ADA biotin-Humira® complexes were then captured on streptavidin-coated plates, washed, and the ADA released into solution using 100 mM acetic acid. The ADA enriched sample was neutralized with 150 mM Tris buffer and analyzed in the multiplexed assay

    Techniques Used: Labeling

    4) Product Images from "Control of VWF A2 domain stability and ADAMTS13 access to the scissile bond of full-length VWF"

    Article Title: Control of VWF A2 domain stability and ADAMTS13 access to the scissile bond of full-length VWF

    Journal: Blood

    doi: 10.1182/blood-2013-11-538173

    Detection of proteolysis of FL-VWF variants by collagen-binding assay. (A-D) The VWF variants and ADAMTS13 were separately preincubated in 20 mM Tris (pH 7.8), 50 mM NaCl, 5 mM CaCl 2 ± 1.5 M urea at 37°C for 45 minutes. VWF (1 µg/mL)
    Figure Legend Snippet: Detection of proteolysis of FL-VWF variants by collagen-binding assay. (A-D) The VWF variants and ADAMTS13 were separately preincubated in 20 mM Tris (pH 7.8), 50 mM NaCl, 5 mM CaCl 2 ± 1.5 M urea at 37°C for 45 minutes. VWF (1 µg/mL)

    Techniques Used: Binding Assay

    5) Product Images from "Plant cell wall glycosyltransferases: High-throughput recombinant expression screening and general requirements for these challenging enzymes"

    Article Title: Plant cell wall glycosyltransferases: High-throughput recombinant expression screening and general requirements for these challenging enzymes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0177591

    The effect of salts, pH and additives in the lysis buffer on the solubility of RGP1. Western blots against a cloning scar shows the varying amount of RGP1 in the soluble lysis fraction after lysing in different buffers. The arrow indicates the band corresponding to full-length RGP1 (42 kDa). Buffers 13 and 19–21, containing 1-2M salt, as well as the low-pH (4.3–5.5) buffers 11,12,15, 22 and 30 decrease the amount of soluble RGP1, while lower salt concentrations as well as several additives increase the amount of soluble RGP1 relative to the standard lysis buffer (std: 50 mM HEPES pH 8.0, 250 mM NaCl, 5 mM MgCl 2 ). MSG = monosodium glutamate, Dex = dextran. *Condition 24: 100 mM triethanolamine, 80 mM sodium glutamate, 0.02% n-octyl-β-D-glucoside, 10% glycerol, 5mM MgSO 4 , pH 8.5. **Condition 29: 100 mM Tris, 100 mM KCl, 0.1% deoxycholate, 25% glycerol, pH 7.6. ***Condition 30: 100 mM potassium acetate, 50 mM NaCl, 0.05% dextran sulfate, 0.1% CHAPS, pH 5.5.
    Figure Legend Snippet: The effect of salts, pH and additives in the lysis buffer on the solubility of RGP1. Western blots against a cloning scar shows the varying amount of RGP1 in the soluble lysis fraction after lysing in different buffers. The arrow indicates the band corresponding to full-length RGP1 (42 kDa). Buffers 13 and 19–21, containing 1-2M salt, as well as the low-pH (4.3–5.5) buffers 11,12,15, 22 and 30 decrease the amount of soluble RGP1, while lower salt concentrations as well as several additives increase the amount of soluble RGP1 relative to the standard lysis buffer (std: 50 mM HEPES pH 8.0, 250 mM NaCl, 5 mM MgCl 2 ). MSG = monosodium glutamate, Dex = dextran. *Condition 24: 100 mM triethanolamine, 80 mM sodium glutamate, 0.02% n-octyl-β-D-glucoside, 10% glycerol, 5mM MgSO 4 , pH 8.5. **Condition 29: 100 mM Tris, 100 mM KCl, 0.1% deoxycholate, 25% glycerol, pH 7.6. ***Condition 30: 100 mM potassium acetate, 50 mM NaCl, 0.05% dextran sulfate, 0.1% CHAPS, pH 5.5.

    Techniques Used: Lysis, Solubility, Western Blot, Clone Assay

    6) Product Images from "A common mechanism of proteasome impairment by neurodegenerative disease-associated oligomers"

    Article Title: A common mechanism of proteasome impairment by neurodegenerative disease-associated oligomers

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03509-0

    The A11(+) oligomers bind to the 20S proteasome and impair opening of the substrate gate. a 20S proteasomes (0.4 μg) and pure non-crosslinked Aβ*56 oligomers (1.5 μg) were incubated separately or together for 30 min (37 °C), crosslinked with 1 mM glutaraldehyde for 5 min, and separated by Native-PAGE (4–8% Tris–acetate gel). Total protein was detected by silver stain (left), and total Aβ was detected by western blot (right). b – d The activity of yeast 20S wild-type (WT) and open-gate (α3ΔN) proteasomes was measured for all three proteolytic sites in the presence of A11(+) oligomers from Aβ*56 ( b ; 2.5 μM), α-Syn ( c ; 100 nM), and Htt-53Q ( d ; 50 nM). Chymotrypsin-like activity was measured by LLVY-amc hydrolysis, trypsin-like activity by RLR-amc, and caspase like by nLPnLD-amc hydrolysis. The concentrations of aggregates are calculated based on the respective monomeric peptide/protein mass. All controls contained an equal volume of buffer identical to that of the respective aggregates. The data are representative of three or more independent experiments performed in triplicate. Error bars represent ± standard deviation
    Figure Legend Snippet: The A11(+) oligomers bind to the 20S proteasome and impair opening of the substrate gate. a 20S proteasomes (0.4 μg) and pure non-crosslinked Aβ*56 oligomers (1.5 μg) were incubated separately or together for 30 min (37 °C), crosslinked with 1 mM glutaraldehyde for 5 min, and separated by Native-PAGE (4–8% Tris–acetate gel). Total protein was detected by silver stain (left), and total Aβ was detected by western blot (right). b – d The activity of yeast 20S wild-type (WT) and open-gate (α3ΔN) proteasomes was measured for all three proteolytic sites in the presence of A11(+) oligomers from Aβ*56 ( b ; 2.5 μM), α-Syn ( c ; 100 nM), and Htt-53Q ( d ; 50 nM). Chymotrypsin-like activity was measured by LLVY-amc hydrolysis, trypsin-like activity by RLR-amc, and caspase like by nLPnLD-amc hydrolysis. The concentrations of aggregates are calculated based on the respective monomeric peptide/protein mass. All controls contained an equal volume of buffer identical to that of the respective aggregates. The data are representative of three or more independent experiments performed in triplicate. Error bars represent ± standard deviation

    Techniques Used: Incubation, Clear Native PAGE, Silver Staining, Western Blot, Activity Assay, Standard Deviation

    7) Product Images from "Monodisperse 130 kDa and 260 kDa Recombinant Human Hemoglobin Polymers as Scaffolds for Protein Engineering of Hemoglobin-Based Oxygen Carriers"

    Article Title: Monodisperse 130 kDa and 260 kDa Recombinant Human Hemoglobin Polymers as Scaffolds for Protein Engineering of Hemoglobin-Based Oxygen Carriers

    Journal: Journal of Functional Biomaterials

    doi: 10.3390/jfb3010061

    Tris-glycine gels (4%–20%) stained with coomassie brilliant blue. ( a ) SDS-PAGE of SGE 953 and rHb1.1. Lane 1, MW markers. MWs in kDa are indicated. Lane 2, partially purified dihemoglobin from SGE 953. This material is the collected fraction from the first chromatography step. Lane 3, fully purified dihemoglobin from SGE 953. Lane 4, purified rHb1.1; ( b ) SDS-PAGE of rHb1.1, rHb1.1 K158C, SGE 953 and SGE 2812 before and after chemical crosslinking; ( c ) Native PAGE of rHb1.1, rHb1.1 K158C, SGE 953, SGE 2812 before and after chemical crosslinking, and a glutaraldehyde-crosslinked “penta-Hb”.
    Figure Legend Snippet: Tris-glycine gels (4%–20%) stained with coomassie brilliant blue. ( a ) SDS-PAGE of SGE 953 and rHb1.1. Lane 1, MW markers. MWs in kDa are indicated. Lane 2, partially purified dihemoglobin from SGE 953. This material is the collected fraction from the first chromatography step. Lane 3, fully purified dihemoglobin from SGE 953. Lane 4, purified rHb1.1; ( b ) SDS-PAGE of rHb1.1, rHb1.1 K158C, SGE 953 and SGE 2812 before and after chemical crosslinking; ( c ) Native PAGE of rHb1.1, rHb1.1 K158C, SGE 953, SGE 2812 before and after chemical crosslinking, and a glutaraldehyde-crosslinked “penta-Hb”.

    Techniques Used: Staining, SDS Page, Purification, Chromatography, Clear Native PAGE

    8) Product Images from "The conformation of the histone H3 tail inhibits association of the BPTF PHD finger with the nucleosome"

    Article Title: The conformation of the histone H3 tail inhibits association of the BPTF PHD finger with the nucleosome

    Journal: eLife

    doi: 10.7554/eLife.31481

    Gel characterization of NCP samples. ( A ) A representative 18% SDS-PAGE gel on NCP samples is shown. The 18% tris gel was run in Tris/glycine/SDS running buffer. Spectra Multicolor Broad Range Protein Ladder (ThermoFisher Scientific) was run alongside NCP samples. The histones are labeled. The H3K C 4me3/4xK-Q and H3K C 4me3/3xR-A histone mutants migrate faster on the gel (likely due to the change in charge composition) and are not resolved from H2A. A small amount of high molecular weight contaminants from the histone preps is visible and marked (*). Additionally, a representative 5% 75:1 acrylamide/bisacrylamide native gel run is shown. The gel was made and run in 0.2x TBE with TrackIt 100 bp DNA ladder (ThermoFisher Scientific) for reference. The gel was run on ice for 50 min at 125V and visualized via ethidium bromide staining. The NCP prepared with different H3 mutants and modifications are not distinguishable under these conditions. A small amount of free 601 DNA (which stains better than DNA within NCP) always remains after the sucrose gradient purification. ( B ). Native gels run on different NCP samples before and after NMR titrations show that the NCP remains intact over the course of the experiments. Note that different amounts of NCP were loaded on the left-most gel. Equal amounts of NCP were loaded before and after the NMR titration for the unmodified NCP (center gel). A denaturing gel run with the unmodified NCP shows that the histones were not degraded over the course of the NMR titration. ( C ). An 18% SDS-PAGE gel was prepared with 50 μM Zn 2+ -Phos-tag acrylamide AAL-107 in order to assess the degree of phosphorylation by AurB. The Spectra BR ladder is simply provided for reference; proteins are known to run slower and differently using Phos-tag acrylamide such that molecular weight estimation is no longer possible. The Phos-tag gel appears to smear and accentuate the high molecular contaminants (*), which might also be due to the 601 DNA present in the sample and running at a similar position as the contaminants. See the comparison with the standard 18% SDS-PAGE gel in ( A ). The gel indicates that the majority of the H3 component was phosphorylated by AurB in the H3K C 4me3/S10ph/S28ph-NCP sample. The different NCP samples are numbered as shown in the legend.
    Figure Legend Snippet: Gel characterization of NCP samples. ( A ) A representative 18% SDS-PAGE gel on NCP samples is shown. The 18% tris gel was run in Tris/glycine/SDS running buffer. Spectra Multicolor Broad Range Protein Ladder (ThermoFisher Scientific) was run alongside NCP samples. The histones are labeled. The H3K C 4me3/4xK-Q and H3K C 4me3/3xR-A histone mutants migrate faster on the gel (likely due to the change in charge composition) and are not resolved from H2A. A small amount of high molecular weight contaminants from the histone preps is visible and marked (*). Additionally, a representative 5% 75:1 acrylamide/bisacrylamide native gel run is shown. The gel was made and run in 0.2x TBE with TrackIt 100 bp DNA ladder (ThermoFisher Scientific) for reference. The gel was run on ice for 50 min at 125V and visualized via ethidium bromide staining. The NCP prepared with different H3 mutants and modifications are not distinguishable under these conditions. A small amount of free 601 DNA (which stains better than DNA within NCP) always remains after the sucrose gradient purification. ( B ). Native gels run on different NCP samples before and after NMR titrations show that the NCP remains intact over the course of the experiments. Note that different amounts of NCP were loaded on the left-most gel. Equal amounts of NCP were loaded before and after the NMR titration for the unmodified NCP (center gel). A denaturing gel run with the unmodified NCP shows that the histones were not degraded over the course of the NMR titration. ( C ). An 18% SDS-PAGE gel was prepared with 50 μM Zn 2+ -Phos-tag acrylamide AAL-107 in order to assess the degree of phosphorylation by AurB. The Spectra BR ladder is simply provided for reference; proteins are known to run slower and differently using Phos-tag acrylamide such that molecular weight estimation is no longer possible. The Phos-tag gel appears to smear and accentuate the high molecular contaminants (*), which might also be due to the 601 DNA present in the sample and running at a similar position as the contaminants. See the comparison with the standard 18% SDS-PAGE gel in ( A ). The gel indicates that the majority of the H3 component was phosphorylated by AurB in the H3K C 4me3/S10ph/S28ph-NCP sample. The different NCP samples are numbered as shown in the legend.

    Techniques Used: SDS Page, Labeling, Molecular Weight, Staining, Purification, Nuclear Magnetic Resonance, Titration

    9) Product Images from "Intrinsic Buffer Hydroxyl Radical Dosimetry Using Tris(Hydroxymethyl)Aminomethane"

    Article Title: Intrinsic Buffer Hydroxyl Radical Dosimetry Using Tris(Hydroxymethyl)Aminomethane

    Journal: bioRxiv

    doi: 10.1101/825463

    Absorbance of Tris at 265 nm as measured by NanoDrop after exposure to four sample conditions. Absorbance increases significantly only upon reaction with hydroxyl radicals generated by flash photolysis of hydrogen peroxide.
    Figure Legend Snippet: Absorbance of Tris at 265 nm as measured by NanoDrop after exposure to four sample conditions. Absorbance increases significantly only upon reaction with hydroxyl radicals generated by flash photolysis of hydrogen peroxide.

    Techniques Used: Generated

    10) Product Images from "Using pyrene-labeled HIV-1 TAR to measure RNA-small molecule binding"

    Article Title: Using pyrene-labeled HIV-1 TAR to measure RNA-small molecule binding

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkg755

    ( A ) The fluorescence spectrum of TAR-2′pyr(25) in the absence and presence of purified neomycin B, in 50 mM Tris pH 7.4, 1 mM MgCl 2 and 100 mM NaCl. The dotted line indicates the fluorescence spectrum of the buffer alone. This spectrum, with an excitation wavelength of 340 nm and slit widths of 12 nm for excitation and 10 nm for emission, has a λ max of 390 nm. ( B ) The fluorescence spectrum of a 5′-U-(2′-amino- butyryl-pyrene-uridine)-U 3mer in the presence and absence of purified neomycin B, in 50 mM Tris pH 7.4, 1 mM MgCl 2 and 100 mM NaCl. The slight decrease in intensity reflects dilution. ( C ), after correcting for dilution and for the residual fluorescence of neomycin B at higher concentrations (shown in filled squares). The open squares demonstrate that the fluorescence of a U-(2′-amino-butyryl-pyrene-uridine)-U 3mer does not increase over this range of neomycin concentrations. Error bars indicate the standard deviation of at least three independent measurements at each aminoglycoside concentration.
    Figure Legend Snippet: ( A ) The fluorescence spectrum of TAR-2′pyr(25) in the absence and presence of purified neomycin B, in 50 mM Tris pH 7.4, 1 mM MgCl 2 and 100 mM NaCl. The dotted line indicates the fluorescence spectrum of the buffer alone. This spectrum, with an excitation wavelength of 340 nm and slit widths of 12 nm for excitation and 10 nm for emission, has a λ max of 390 nm. ( B ) The fluorescence spectrum of a 5′-U-(2′-amino- butyryl-pyrene-uridine)-U 3mer in the presence and absence of purified neomycin B, in 50 mM Tris pH 7.4, 1 mM MgCl 2 and 100 mM NaCl. The slight decrease in intensity reflects dilution. ( C ), after correcting for dilution and for the residual fluorescence of neomycin B at higher concentrations (shown in filled squares). The open squares demonstrate that the fluorescence of a U-(2′-amino-butyryl-pyrene-uridine)-U 3mer does not increase over this range of neomycin concentrations. Error bars indicate the standard deviation of at least three independent measurements at each aminoglycoside concentration.

    Techniques Used: Fluorescence, Purification, Standard Deviation, Concentration Assay

    11) Product Images from "Manipulating Ionic Strength to Improve Single Cell Electrophoretic Separations"

    Article Title: Manipulating Ionic Strength to Improve Single Cell Electrophoretic Separations

    Journal: Talanta

    doi: 10.1016/j.talanta.2013.03.012

    Summary of conditions used in the analysis of GSL metabolism in single dPC12 cells. A) A single dPC12 cell, suspended in PBS and bracketed between two plugs of 1% SDS in PBS, was separated using a 100 mM TRIS, 100 mM CHES, 20 mM SDS, and 5 mM α-CD
    Figure Legend Snippet: Summary of conditions used in the analysis of GSL metabolism in single dPC12 cells. A) A single dPC12 cell, suspended in PBS and bracketed between two plugs of 1% SDS in PBS, was separated using a 100 mM TRIS, 100 mM CHES, 20 mM SDS, and 5 mM α-CD

    Techniques Used:

    12) Product Images from "Transmission electron microscopy characterization of fluorescently labelled amyloid ? 1-40 and ?-synuclein aggregates"

    Article Title: Transmission electron microscopy characterization of fluorescently labelled amyloid ? 1-40 and ?-synuclein aggregates

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-11-125

    TEM images of aggregates grown from seeded or unfiltered αS-EGFP stocks . The scale bar is 200 nm wide and all images are shown at the same magnification. Unless otherwise specified, all solutions contained PBS buffer (10 mM pH 7.5 NaPhos, 150 mM NaCl) and the samples were incubated with shaking at 37°C. The protein concentrations, incubation times t , and other solution conditions are as follows: (A-B) 75 μM unfiltered αS-EGFP, t = 3 weeks. (C) 150 μM unfiltered αS-EGFP, t = 3 weeks. (D) 35 μM unfiltered αS-EGFP in pH 7.4 Tris buffer with 100 mM NaCl, t = 3 weeks. (E-G) 30 μM filtered αS-EGFP plus ~4 μM unfiltered αS-EGFP
    Figure Legend Snippet: TEM images of aggregates grown from seeded or unfiltered αS-EGFP stocks . The scale bar is 200 nm wide and all images are shown at the same magnification. Unless otherwise specified, all solutions contained PBS buffer (10 mM pH 7.5 NaPhos, 150 mM NaCl) and the samples were incubated with shaking at 37°C. The protein concentrations, incubation times t , and other solution conditions are as follows: (A-B) 75 μM unfiltered αS-EGFP, t = 3 weeks. (C) 150 μM unfiltered αS-EGFP, t = 3 weeks. (D) 35 μM unfiltered αS-EGFP in pH 7.4 Tris buffer with 100 mM NaCl, t = 3 weeks. (E-G) 30 μM filtered αS-EGFP plus ~4 μM unfiltered αS-EGFP "seed" in pH 7.4 Tris with 100 mM NaCl, t = 4 weeks. (H) 20 μM unfiltered αS-EGFP in pH 7.4 Tris with 100 mM NaCl, t = 4 weeks. (I-J) 150 μM filtered αS-EGFP plus ~ 8 μM unfiltered αS-EGFP "seed", t = 4 weeks.

    Techniques Used: Transmission Electron Microscopy, Incubation

    13) Product Images from "Immuno- and Constitutive Proteasomes Do Not Differ in Their Abilities to Degrade Ubiquitinated Proteins"

    Article Title: Immuno- and Constitutive Proteasomes Do Not Differ in Their Abilities to Degrade Ubiquitinated Proteins

    Journal: Cell

    doi: 10.1016/j.cell.2013.01.037

    IFNγ Treatment Does Not Cause a Significant Accumulation of Polyubiquitin Conjugates during the Induction of Immunoproteasomes, Related to Figure 1 (A and B) Murine embryonic (A) and B8 (B) fibroblasts were stimulated with 100U/ml IFNγ for the indicated time periods. As a control for ubiquitin accumulation, cells were treated with the proteasome inhibitor lactacystin (10μM, 4hr) or vehicle (DMSO). The cells were then lysed by sonication in 25mM HEPES pH 7.4, 1mM ATP, 1mM DTT, 5mM MgCl 2 and 10% glycerol. Lysates were subsequently centrifuged for one hour at 100,000 x g. Protein concentrations of the lysates were determined using the DC Protein Assay (Biorad) and equal amounts of protein were separated by SDS-PAGE and analyzed by western blotting. α-tubulin served as a loading control. One representative experiment out of three independent experiments with similar outcomes is shown. The induction of LMP7 by IFNγ stimulation was confirmed by immunoblot analysis; GAPDH served as a loading control. The ubiquitin levels were determined by densitometric analyses (ImageJ) of five different western blots; shown are the mean values ± SD obtained after normalization to the loading control and relative to the value for unstimulated cells which was set to unity. (C and D) HeLa cells were treated with 100U/ml IFNγ as described (C) or with 10 μM lactacystin for 4 hr (D). The cells were lysed with nonionic detergent according to the methods described by Seifert et al. (20 mM TRIS-HCl, pH 7.5, 10 mM EDTA, 100 mM NaCl, 1% NP40, 10 μM MG-132, 5 mM NEM and Complete Protease Inhibitor Cocktail (Roche)) and immunblotted for ubiquitin (C, left). The ubiquitin levels, (relative to the β-actin control) were determined by densitometric analyses (ImageJ) as described (C, right). The mean values ± SEM for three replicates are shown (C).
    Figure Legend Snippet: IFNγ Treatment Does Not Cause a Significant Accumulation of Polyubiquitin Conjugates during the Induction of Immunoproteasomes, Related to Figure 1 (A and B) Murine embryonic (A) and B8 (B) fibroblasts were stimulated with 100U/ml IFNγ for the indicated time periods. As a control for ubiquitin accumulation, cells were treated with the proteasome inhibitor lactacystin (10μM, 4hr) or vehicle (DMSO). The cells were then lysed by sonication in 25mM HEPES pH 7.4, 1mM ATP, 1mM DTT, 5mM MgCl 2 and 10% glycerol. Lysates were subsequently centrifuged for one hour at 100,000 x g. Protein concentrations of the lysates were determined using the DC Protein Assay (Biorad) and equal amounts of protein were separated by SDS-PAGE and analyzed by western blotting. α-tubulin served as a loading control. One representative experiment out of three independent experiments with similar outcomes is shown. The induction of LMP7 by IFNγ stimulation was confirmed by immunoblot analysis; GAPDH served as a loading control. The ubiquitin levels were determined by densitometric analyses (ImageJ) of five different western blots; shown are the mean values ± SD obtained after normalization to the loading control and relative to the value for unstimulated cells which was set to unity. (C and D) HeLa cells were treated with 100U/ml IFNγ as described (C) or with 10 μM lactacystin for 4 hr (D). The cells were lysed with nonionic detergent according to the methods described by Seifert et al. (20 mM TRIS-HCl, pH 7.5, 10 mM EDTA, 100 mM NaCl, 1% NP40, 10 μM MG-132, 5 mM NEM and Complete Protease Inhibitor Cocktail (Roche)) and immunblotted for ubiquitin (C, left). The ubiquitin levels, (relative to the β-actin control) were determined by densitometric analyses (ImageJ) as described (C, right). The mean values ± SEM for three replicates are shown (C).

    Techniques Used: Sonication, DC Protein Assay, SDS Page, Western Blot, Protease Inhibitor

    14) Product Images from "Phase separation directs ubiquitination of gene body nucleosomes"

    Article Title: Phase separation directs ubiquitination of gene body nucleosomes

    Journal: Nature

    doi: 10.1038/s41586-020-2097-z

    Specific features of yeast Lge1 and human WAC contribute to H2BK123ub and cell viability. a . The WAC and LAF1 IDRs promote LLPS. Phase-separation assay of recombinant 6His-Lge1 and the fusion constructs 6His-WAC(1-318)-Lge1 CC and 6His-LAF1(1-169)-Lge1 CC (both proteins 5 μM; buffer 20 mM Tris pH 7.5, 100 mM NaCl, 1 mM DTT, 20 °C). Scale bar, 10 μm. Protein inputs are shown on the right (black arrows), b . Quantification of condensate sizes (μm 2 ) in a . n = number of condensates. Dot plot showing median and interquartile range. **p-value = 0.004, ****p-value
    Figure Legend Snippet: Specific features of yeast Lge1 and human WAC contribute to H2BK123ub and cell viability. a . The WAC and LAF1 IDRs promote LLPS. Phase-separation assay of recombinant 6His-Lge1 and the fusion constructs 6His-WAC(1-318)-Lge1 CC and 6His-LAF1(1-169)-Lge1 CC (both proteins 5 μM; buffer 20 mM Tris pH 7.5, 100 mM NaCl, 1 mM DTT, 20 °C). Scale bar, 10 μm. Protein inputs are shown on the right (black arrows), b . Quantification of condensate sizes (μm 2 ) in a . n = number of condensates. Dot plot showing median and interquartile range. **p-value = 0.004, ****p-value

    Techniques Used: Recombinant, Construct

    15) Product Images from "Characterization of Nora Virus Structural Proteins via Western Blot Analysis"

    Article Title: Characterization of Nora Virus Structural Proteins via Western Blot Analysis

    Journal: Scientifica

    doi: 10.1155/2016/9067848

    Two distinct sized particles are produced from a Nora virus infection of Drosophila melanogaster witi Rel E23 strain flies. Approximately 3,000 Nora virus infected flies were homogenized and processed as described in Section 2 . In (a) a representative CsCl gradient is shown, depicting the two bands of virus particles obtained. The bands were collected individually and dialyzed against 10 mM Tris-HCl, pH 8.0, and 250 mM NaCl buffer. Electron micrographs of the particles obtained from the upper band (b) and the lower band (c) are shown. The average sizes of the virus particles were 31.8 nm (upper band; n = 47) and 29.7 nm (lower band; n = 39), respectively.
    Figure Legend Snippet: Two distinct sized particles are produced from a Nora virus infection of Drosophila melanogaster witi Rel E23 strain flies. Approximately 3,000 Nora virus infected flies were homogenized and processed as described in Section 2 . In (a) a representative CsCl gradient is shown, depicting the two bands of virus particles obtained. The bands were collected individually and dialyzed against 10 mM Tris-HCl, pH 8.0, and 250 mM NaCl buffer. Electron micrographs of the particles obtained from the upper band (b) and the lower band (c) are shown. The average sizes of the virus particles were 31.8 nm (upper band; n = 47) and 29.7 nm (lower band; n = 39), respectively.

    Techniques Used: Produced, Infection

    16) Product Images from "SHAPE analysis of the FIV Leader RNA reveals a structural switch potentially controlling viral packaging and genome dimerization"

    Article Title: SHAPE analysis of the FIV Leader RNA reveals a structural switch potentially controlling viral packaging and genome dimerization

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr252

    Dimerization analysis of SL5 mutants. ( A ) Dimerization assay and 1M7 reactivity of SL5 mutants. In vitro transcribed FIV packaging signal RNAs containing a wt or mutant SL5 sequence were heated to 95°C, snap-cooled, and incubated at 55°C for 4 h in 10 mM Tris–HCl, pH 7, 200 mM NaCl, 4 mM MgCl 2 before electrophoresis on an agarose gel in TBM, or SHAPE analysis as described in ‘Materials and Methods’ section. Mutant designations are given above the relevant lanes and their sequences at SL5 are illustrated above or below each lane. L; RNA ladder. Nucleotide colours show 1M7 reactivities as shown on the key. ( B ) Densitometric analysis of dimerization on TBM (black bars) or TBE gels (striped bars). Stars represent statistical significance relative to wt ( P
    Figure Legend Snippet: Dimerization analysis of SL5 mutants. ( A ) Dimerization assay and 1M7 reactivity of SL5 mutants. In vitro transcribed FIV packaging signal RNAs containing a wt or mutant SL5 sequence were heated to 95°C, snap-cooled, and incubated at 55°C for 4 h in 10 mM Tris–HCl, pH 7, 200 mM NaCl, 4 mM MgCl 2 before electrophoresis on an agarose gel in TBM, or SHAPE analysis as described in ‘Materials and Methods’ section. Mutant designations are given above the relevant lanes and their sequences at SL5 are illustrated above or below each lane. L; RNA ladder. Nucleotide colours show 1M7 reactivities as shown on the key. ( B ) Densitometric analysis of dimerization on TBM (black bars) or TBE gels (striped bars). Stars represent statistical significance relative to wt ( P

    Techniques Used: In Vitro, Mutagenesis, Sequencing, Incubation, Electrophoresis, Agarose Gel Electrophoresis

    17) Product Images from "Pseudomonas aeruginosa elastase cleaves a C-terminal peptide from human thrombin that inhibits host inflammatory responses"

    Article Title: Pseudomonas aeruginosa elastase cleaves a C-terminal peptide from human thrombin that inhibits host inflammatory responses

    Journal: Nature Communications

    doi: 10.1038/ncomms11567

    In vivo relevance of FYT21. ( a ) Wound fluids from non-healing leg ulcers (numbers 1–5) were analysed by western blotting using an antibody recognizing the C-terminal part of thrombin; plasma (p) is shown in lane 2. ( b ) An EM picture of a leukocyte, located in fibrin slough from an infected chronic wound, containing C-terminal epitopes of thrombin (black dots). The scale bars indicate 2 μm (left) and 100 nm (right). ( c ) Representative LC–MS/MS chromatogram of affinity purified wound dressing extract depicted in black; HVF18 eluted at 54 min and FYT21 at 59 min. The Orbitrap mass spectra of the triple-charged HVF18 peptide ( m / z 757.7625) and the quadruple-charged FYT21 peptide ( m / z 671.3670) enabled mass determination of both peptides (isotopic distribution depicted in grey). The assigned fragmentation pattern of HVF18 is given in red. ( d ) Cytokine analysis of blood collected from Balb/c mice (female 10–12 weeks), 4 h after i.p. injection with 1 mg kg −1 of LPS or PBS, which was followed after 30 min with injection of FYT21 (500 μg) or 10 mM Tris, pH 7.4. Control mice were injected twice with buffer. Results are means±s.e.m. of three independent experiments with 3–6 mice per group. ( e ) In vivo image of mice injected s.c. with the reactive oxygen species reporter probe L-012, 4 h after i.p. injection with 5 mg kg −1 of LPS and 500 μg FYT21 as described in d . Results are means±s.e.m. of three independent experiments with 3 mice per group. ( f ) A representative image of the four groups at 2 h and 4 h after injection: C, control; FYT, FYT21; L+F, LPS+FYT21. Values are significantly (* P
    Figure Legend Snippet: In vivo relevance of FYT21. ( a ) Wound fluids from non-healing leg ulcers (numbers 1–5) were analysed by western blotting using an antibody recognizing the C-terminal part of thrombin; plasma (p) is shown in lane 2. ( b ) An EM picture of a leukocyte, located in fibrin slough from an infected chronic wound, containing C-terminal epitopes of thrombin (black dots). The scale bars indicate 2 μm (left) and 100 nm (right). ( c ) Representative LC–MS/MS chromatogram of affinity purified wound dressing extract depicted in black; HVF18 eluted at 54 min and FYT21 at 59 min. The Orbitrap mass spectra of the triple-charged HVF18 peptide ( m / z 757.7625) and the quadruple-charged FYT21 peptide ( m / z 671.3670) enabled mass determination of both peptides (isotopic distribution depicted in grey). The assigned fragmentation pattern of HVF18 is given in red. ( d ) Cytokine analysis of blood collected from Balb/c mice (female 10–12 weeks), 4 h after i.p. injection with 1 mg kg −1 of LPS or PBS, which was followed after 30 min with injection of FYT21 (500 μg) or 10 mM Tris, pH 7.4. Control mice were injected twice with buffer. Results are means±s.e.m. of three independent experiments with 3–6 mice per group. ( e ) In vivo image of mice injected s.c. with the reactive oxygen species reporter probe L-012, 4 h after i.p. injection with 5 mg kg −1 of LPS and 500 μg FYT21 as described in d . Results are means±s.e.m. of three independent experiments with 3 mice per group. ( f ) A representative image of the four groups at 2 h and 4 h after injection: C, control; FYT, FYT21; L+F, LPS+FYT21. Values are significantly (* P

    Techniques Used: In Vivo, Western Blot, Infection, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Affinity Purification, Mouse Assay, Injection

    18) Product Images from "EhMAPK, the Mitogen-Activated Protein Kinase from Entamoeba histolytica Is Associated with Cell Survival"

    Article Title: EhMAPK, the Mitogen-Activated Protein Kinase from Entamoeba histolytica Is Associated with Cell Survival

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0013291

    Expression, purification and antibody production of recombinant EhMAPK. (a) Western blot analysis with mouse anti-His antibody to check the expression of recombinant His tagged EhMAPK. Lane1: uninduced E. coli BL21DE3 cell lysate, Lane 2: Cell lysate from E. coli BL21DE3 induced with 1 mM IPTG at 37°C for 3 h. (b) Coomassie stained gel showing the purification of recombinant His tagged EhMAPK protein with Ni-NTA affinity chromatography. Lane 1: Protein molecular weight marker (Fermentas), Lane 2: Uninduced transformed E. coli BL21DE3 cell lysate, Lane 3: Cell lysate from transformed E. coli BL21DE3 induced with 1 mM IPTG at 37°C for 3 h, Lane 4: Inclusion body solubilised in 50 mM Tris pH 8, 300 mM NaCl, 8 M urea and 10 mM imidazole, Lane 5: Purified recombinant EhMAPK. (c) Western immunoblotting with post and pre immune sera from rabbits immunized with purified recombinant EhMAPK. Lane 1–3: 1 µg of purified recombinant EhMAPK, 20 µg and 40 µg of E. histolytica total cell lysate (TCL) respectively probed with post immune rabbit sera, Lane 4–6: 1 µg of purified recombinant EhMAPK, 20 µg and 40 µg of E. histolytica TCL respectively probed with pre immune rabbit sera.
    Figure Legend Snippet: Expression, purification and antibody production of recombinant EhMAPK. (a) Western blot analysis with mouse anti-His antibody to check the expression of recombinant His tagged EhMAPK. Lane1: uninduced E. coli BL21DE3 cell lysate, Lane 2: Cell lysate from E. coli BL21DE3 induced with 1 mM IPTG at 37°C for 3 h. (b) Coomassie stained gel showing the purification of recombinant His tagged EhMAPK protein with Ni-NTA affinity chromatography. Lane 1: Protein molecular weight marker (Fermentas), Lane 2: Uninduced transformed E. coli BL21DE3 cell lysate, Lane 3: Cell lysate from transformed E. coli BL21DE3 induced with 1 mM IPTG at 37°C for 3 h, Lane 4: Inclusion body solubilised in 50 mM Tris pH 8, 300 mM NaCl, 8 M urea and 10 mM imidazole, Lane 5: Purified recombinant EhMAPK. (c) Western immunoblotting with post and pre immune sera from rabbits immunized with purified recombinant EhMAPK. Lane 1–3: 1 µg of purified recombinant EhMAPK, 20 µg and 40 µg of E. histolytica total cell lysate (TCL) respectively probed with post immune rabbit sera, Lane 4–6: 1 µg of purified recombinant EhMAPK, 20 µg and 40 µg of E. histolytica TCL respectively probed with pre immune rabbit sera.

    Techniques Used: Expressing, Purification, Recombinant, Western Blot, Staining, Affinity Chromatography, Molecular Weight, Marker, Transformation Assay

    19) Product Images from "A common mechanism of proteasome impairment by neurodegenerative disease-associated oligomers"

    Article Title: A common mechanism of proteasome impairment by neurodegenerative disease-associated oligomers

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03509-0

    The A11(+) oligomers bind to the 20S proteasome and impair opening of the substrate gate. a 20S proteasomes (0.4 μg) and pure non-crosslinked Aβ*56 oligomers (1.5 μg) were incubated separately or together for 30 min (37 °C), crosslinked with 1 mM glutaraldehyde for 5 min, and separated by Native-PAGE (4–8% Tris–acetate gel). Total protein was detected by silver stain (left), and total Aβ was detected by western blot (right). b – d The activity of yeast 20S wild-type (WT) and open-gate (α3ΔN) proteasomes was measured for all three proteolytic sites in the presence of A11(+) oligomers from Aβ*56 ( b ; 2.5 μM), α-Syn ( c ; 100 nM), and Htt-53Q ( d ; 50 nM). Chymotrypsin-like activity was measured by LLVY-amc hydrolysis, trypsin-like activity by RLR-amc, and caspase like by nLPnLD-amc hydrolysis. The concentrations of aggregates are calculated based on the respective monomeric peptide/protein mass. All controls contained an equal volume of buffer identical to that of the respective aggregates. The data are representative of three or more independent experiments performed in triplicate. Error bars represent ± standard deviation
    Figure Legend Snippet: The A11(+) oligomers bind to the 20S proteasome and impair opening of the substrate gate. a 20S proteasomes (0.4 μg) and pure non-crosslinked Aβ*56 oligomers (1.5 μg) were incubated separately or together for 30 min (37 °C), crosslinked with 1 mM glutaraldehyde for 5 min, and separated by Native-PAGE (4–8% Tris–acetate gel). Total protein was detected by silver stain (left), and total Aβ was detected by western blot (right). b – d The activity of yeast 20S wild-type (WT) and open-gate (α3ΔN) proteasomes was measured for all three proteolytic sites in the presence of A11(+) oligomers from Aβ*56 ( b ; 2.5 μM), α-Syn ( c ; 100 nM), and Htt-53Q ( d ; 50 nM). Chymotrypsin-like activity was measured by LLVY-amc hydrolysis, trypsin-like activity by RLR-amc, and caspase like by nLPnLD-amc hydrolysis. The concentrations of aggregates are calculated based on the respective monomeric peptide/protein mass. All controls contained an equal volume of buffer identical to that of the respective aggregates. The data are representative of three or more independent experiments performed in triplicate. Error bars represent ± standard deviation

    Techniques Used: Incubation, Clear Native PAGE, Silver Staining, Western Blot, Activity Assay, Standard Deviation

    20) Product Images from "Isolation of Monoclonal Antibodies with Predetermined Conformational Epitope Specificity"

    Article Title: Isolation of Monoclonal Antibodies with Predetermined Conformational Epitope Specificity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0038943

    Properties of mAbs 33B2 and 33C6. (A) Binding to envelope proteins of different HIV clades. Plates were coated with Env proteins and probed with newly isolated mAbs along with isotype controls (positive control, HGN194 [16] ; negative control, Fm-6 [37] ) and cognate rhesus monkey (RJa-9) serum. HIV Env proteins were derived from the following strains: clade A, UG37; B, BaL and IIIB; C, CN54 and 1157ip (the latter is a simian-human immunodeficiency virus (SHIV) strain encoding an HIV clade C envelope [25] ; D, UG21. SIVmne gp160 was used as negative control. (B) Epitope mapping. Bars show Ab binding by ELISA to consensus HIV clade C peptides representing the V3 loop region. The control peptide represents the scrambled C-terminal sequence of SHIV-1157ip gp120. (C) Virion binding assay. MAbs 33B2 and 33C6 along with negative control (Fm-6) and positive control (HGN194) were captured on the plate coated with goat anti-human IgG Fc-specific Ab and then exposed to SHIV-1157ipEL-p [19] virions. The amount of virus bound to mAbs was assessed by p27 Gag ELISA. (D) Binding of mAbs 33B2 and 33C6 with native and denatured HIV CN54 gp120. Plates were coated with native, reduced or reduced/denatured HIV Env protein and probed with 33B2 and 33C6 along with Fm-6 (negative control mAb) as well as serum of HIV-positive individual (positive control), and serum of HIV-negative individual (negative control). We used tris (2-carboxyethyl)phosphine (TCEP) to reduce gp120 and TCEP + SDS to reduce and denature, respectively ( Methods ). Each data point represents the mean ± s.e.m. (n = 3). Experiments were repeated 3 times.
    Figure Legend Snippet: Properties of mAbs 33B2 and 33C6. (A) Binding to envelope proteins of different HIV clades. Plates were coated with Env proteins and probed with newly isolated mAbs along with isotype controls (positive control, HGN194 [16] ; negative control, Fm-6 [37] ) and cognate rhesus monkey (RJa-9) serum. HIV Env proteins were derived from the following strains: clade A, UG37; B, BaL and IIIB; C, CN54 and 1157ip (the latter is a simian-human immunodeficiency virus (SHIV) strain encoding an HIV clade C envelope [25] ; D, UG21. SIVmne gp160 was used as negative control. (B) Epitope mapping. Bars show Ab binding by ELISA to consensus HIV clade C peptides representing the V3 loop region. The control peptide represents the scrambled C-terminal sequence of SHIV-1157ip gp120. (C) Virion binding assay. MAbs 33B2 and 33C6 along with negative control (Fm-6) and positive control (HGN194) were captured on the plate coated with goat anti-human IgG Fc-specific Ab and then exposed to SHIV-1157ipEL-p [19] virions. The amount of virus bound to mAbs was assessed by p27 Gag ELISA. (D) Binding of mAbs 33B2 and 33C6 with native and denatured HIV CN54 gp120. Plates were coated with native, reduced or reduced/denatured HIV Env protein and probed with 33B2 and 33C6 along with Fm-6 (negative control mAb) as well as serum of HIV-positive individual (positive control), and serum of HIV-negative individual (negative control). We used tris (2-carboxyethyl)phosphine (TCEP) to reduce gp120 and TCEP + SDS to reduce and denature, respectively ( Methods ). Each data point represents the mean ± s.e.m. (n = 3). Experiments were repeated 3 times.

    Techniques Used: Binding Assay, Isolation, Positive Control, Negative Control, Derivative Assay, Enzyme-linked Immunosorbent Assay, Sequencing

    21) Product Images from "Ribosome profiling reveals pervasive and regulated stop codon readthrough in Drosophila melanogaster"

    Article Title: Ribosome profiling reveals pervasive and regulated stop codon readthrough in Drosophila melanogaster

    Journal: eLife

    doi: 10.7554/eLife.01179

    Digestion with micrococcal nuclease yields a robust ribosome profiling assay. ( A ) Digestion of polysomes with RNase I degrades ribosomes. A lysate was made from S2 cells using a previous version of our protocol. Aliquots of this lysate were digested with increasing amounts of RNase I, and resolved on 10–50% sucrose gradients. As amounts of RNase I increase, the heights of all peaks—including the monosomal (80S) peak—decrease before polysomes are fully resolved to monosomes. ( B ) as in ( A ), but using micrococcal nuclease (MNase) and our current protocol. From 0.5 to 2 U MNase/μg total RNA, monosomes are resolved with no reduction in the size of the monosome peak. This result indicates that Drosophila ribosomes are stable to MNase over a broad range of concentrations, whereas the mRNA between ribosomes is digested. ( C ) Ribosome protection assay. A 320 nucleotide fragment of enolase (FlyBase accession: FBgn0000579) was amplified using oligos oJGD123 oJGD124 ( Supplementary file 2 ). A body-labeled probe against this sequence was transcribed from this template using α32P-UTP and the T7 MaxiScript kit (Ambion). S2 cell lysates were prepared as in methods and aliquoted. Aliquots were digested as in methods, except with 0, 0.5, 1, 2, 3 or 4 U MNase/μg total RNA. Monosomes were sedimented through a sucrose cushion, resuspended in 600 μl 10 mM Tris pH 7.0, and their RNAs extracted as in ‘Materials and methods’. Concentrations were determined using a NanoDrop spectrophotometer. 5 μg of each sample was hybridized to 50,000 CPM of probe overnight at 42°C. Single-stranded regions were digested with RNase A/T1 and the remaining footprint: probe duplexes detected using the mirVana micro-RNA detection kit (Ambion), resolved on a 15% TBE-urea gel (Invitrogen), and visualized on a Storm phosphorimager (Molecular Dynamics by GE Healthcare Bio-Sciences, Pittsburgh, PA). For size markers, we end-labeled the Novex 10 bp dsDNA ladder (Invitrogen) with 32P. Over two-fold range of nuclease concentrations, the ∼30 nt peak corresponding to ribosome-protected footprints remains constant in size and intensity, indicating a lack of degradation consistent with the unchanged monosome peak height across this range of digestion conditions in ( B ). Also visible is a roughly 60 nt band which we infer to be protected by adjacent ribosomes (disomes) that sterically exclude the nuclease. This interpretation is consistent with the presence of a small disome peak in digested samples (c.f. panels B and D , and Figure 1A ). ( D ) A polysome lysate was prepared from S2 cells and resolved in 10–50% sucrose gradients, with or without prior digestion with 3 U MNase/μg total RNA ( E ) A culture of S2 cells was split into aliquots and processed using our current protocol as if they were independent samples. Total counts aligning to the coding region of each gene were tabulated in each replicate. Genes sharing at least 128 footprint counts between replicates (red) are well-correlated, demonstrating the assay is robust (see full discussion in Figure 1—figure supplement 2 ). Source data may be found in supplementary table 1 (at Dryad: Dunn et al., 2013 ). DOI: http://dx.doi.org/10.7554/eLife.01179.004
    Figure Legend Snippet: Digestion with micrococcal nuclease yields a robust ribosome profiling assay. ( A ) Digestion of polysomes with RNase I degrades ribosomes. A lysate was made from S2 cells using a previous version of our protocol. Aliquots of this lysate were digested with increasing amounts of RNase I, and resolved on 10–50% sucrose gradients. As amounts of RNase I increase, the heights of all peaks—including the monosomal (80S) peak—decrease before polysomes are fully resolved to monosomes. ( B ) as in ( A ), but using micrococcal nuclease (MNase) and our current protocol. From 0.5 to 2 U MNase/μg total RNA, monosomes are resolved with no reduction in the size of the monosome peak. This result indicates that Drosophila ribosomes are stable to MNase over a broad range of concentrations, whereas the mRNA between ribosomes is digested. ( C ) Ribosome protection assay. A 320 nucleotide fragment of enolase (FlyBase accession: FBgn0000579) was amplified using oligos oJGD123 oJGD124 ( Supplementary file 2 ). A body-labeled probe against this sequence was transcribed from this template using α32P-UTP and the T7 MaxiScript kit (Ambion). S2 cell lysates were prepared as in methods and aliquoted. Aliquots were digested as in methods, except with 0, 0.5, 1, 2, 3 or 4 U MNase/μg total RNA. Monosomes were sedimented through a sucrose cushion, resuspended in 600 μl 10 mM Tris pH 7.0, and their RNAs extracted as in ‘Materials and methods’. Concentrations were determined using a NanoDrop spectrophotometer. 5 μg of each sample was hybridized to 50,000 CPM of probe overnight at 42°C. Single-stranded regions were digested with RNase A/T1 and the remaining footprint: probe duplexes detected using the mirVana micro-RNA detection kit (Ambion), resolved on a 15% TBE-urea gel (Invitrogen), and visualized on a Storm phosphorimager (Molecular Dynamics by GE Healthcare Bio-Sciences, Pittsburgh, PA). For size markers, we end-labeled the Novex 10 bp dsDNA ladder (Invitrogen) with 32P. Over two-fold range of nuclease concentrations, the ∼30 nt peak corresponding to ribosome-protected footprints remains constant in size and intensity, indicating a lack of degradation consistent with the unchanged monosome peak height across this range of digestion conditions in ( B ). Also visible is a roughly 60 nt band which we infer to be protected by adjacent ribosomes (disomes) that sterically exclude the nuclease. This interpretation is consistent with the presence of a small disome peak in digested samples (c.f. panels B and D , and Figure 1A ). ( D ) A polysome lysate was prepared from S2 cells and resolved in 10–50% sucrose gradients, with or without prior digestion with 3 U MNase/μg total RNA ( E ) A culture of S2 cells was split into aliquots and processed using our current protocol as if they were independent samples. Total counts aligning to the coding region of each gene were tabulated in each replicate. Genes sharing at least 128 footprint counts between replicates (red) are well-correlated, demonstrating the assay is robust (see full discussion in Figure 1—figure supplement 2 ). Source data may be found in supplementary table 1 (at Dryad: Dunn et al., 2013 ). DOI: http://dx.doi.org/10.7554/eLife.01179.004

    Techniques Used: Amplification, Labeling, Sequencing, Spectrophotometry, RNA Detection

    22) Product Images from "Thermostable virus portal proteins as reprogrammable adapters for solid-state nanopore sensors"

    Article Title: Thermostable virus portal proteins as reprogrammable adapters for solid-state nanopore sensors

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07116-x

    Characterization of the hybrid pore formation. a Typical current profile over time recorded through a 5.5 nm diameter SS pore at +100 mV. After injection of 0.1 nmol of portal protein, short current drops are detected, interpreted as portal collisions with the solid-state nanopore. b A representative current vs time trace recorded for a 5.4 nm SS nanopore at +80 mV, showing stable insertion of a portal protein. c Current as a function of the applied voltage for a 5.7 nm diameter SS pore recorded before (red markers) and after insertion of a portal protein (purple). d Current noise analysis of a 5.5 nm diameter solid-state nanopore before (red) and after insertion of a portal protein (purple). e Conductance of solid-state nanopore vs conductance of hybrid SS-portal pores formed after electrokinetic corking of the portal protein within the SS nanopore ( n = 32 for CD/N hybrids and n = 15 for CGG hybrids). Experiments were performed in 0.5 M NaCl, 20 mM Tris, pH 7.5
    Figure Legend Snippet: Characterization of the hybrid pore formation. a Typical current profile over time recorded through a 5.5 nm diameter SS pore at +100 mV. After injection of 0.1 nmol of portal protein, short current drops are detected, interpreted as portal collisions with the solid-state nanopore. b A representative current vs time trace recorded for a 5.4 nm SS nanopore at +80 mV, showing stable insertion of a portal protein. c Current as a function of the applied voltage for a 5.7 nm diameter SS pore recorded before (red markers) and after insertion of a portal protein (purple). d Current noise analysis of a 5.5 nm diameter solid-state nanopore before (red) and after insertion of a portal protein (purple). e Conductance of solid-state nanopore vs conductance of hybrid SS-portal pores formed after electrokinetic corking of the portal protein within the SS nanopore ( n = 32 for CD/N hybrids and n = 15 for CGG hybrids). Experiments were performed in 0.5 M NaCl, 20 mM Tris, pH 7.5

    Techniques Used: Injection

    Sensing different biopolymers using a hybrid nanopore. Current vs time trace recorded through the hybrid pore at +60 mV in the presence of a 36.0 μM insulin, b 7.7 μM DNA hairpin, c 10.3 μM TPX2 peptide and d 16.6 μM ssDNA. The data in a were filtered at 10 kHz (gray) or 0.5 kHz (green). e Scatter plot of Δ I vs dwell time for the DNA hairpin (red, n = 5883 events), the peptide (purple, n = 3368 events) and the ssDNA (orange, n = 18,812 events)). Experiments were performed in 0.5 M NaCl, 20 mM Tris, pH 7.5
    Figure Legend Snippet: Sensing different biopolymers using a hybrid nanopore. Current vs time trace recorded through the hybrid pore at +60 mV in the presence of a 36.0 μM insulin, b 7.7 μM DNA hairpin, c 10.3 μM TPX2 peptide and d 16.6 μM ssDNA. The data in a were filtered at 10 kHz (gray) or 0.5 kHz (green). e Scatter plot of Δ I vs dwell time for the DNA hairpin (red, n = 5883 events), the peptide (purple, n = 3368 events) and the ssDNA (orange, n = 18,812 events)). Experiments were performed in 0.5 M NaCl, 20 mM Tris, pH 7.5

    Techniques Used:

    Dynamics of TPX2 peptide transport. a Current vs time trace recorded through a hybrid pore at +30, +40 and +55 mV in the presence of 10.3 μM TPX2 peptide. b Semi-log plot of the event frequency as a function of the applied voltage. c Semi-log plot of the peptide dwell time as a function of the applied voltage. The lines in ( b , c ) are exponential fits to the data. Experiments were performed in 0.5 M NaCl, 20 mM Tris, pH 7.5. Data shown in semi-log plots are mean and s.d. of 23,516 events (total for all points) from one hybrid nanopore
    Figure Legend Snippet: Dynamics of TPX2 peptide transport. a Current vs time trace recorded through a hybrid pore at +30, +40 and +55 mV in the presence of 10.3 μM TPX2 peptide. b Semi-log plot of the event frequency as a function of the applied voltage. c Semi-log plot of the peptide dwell time as a function of the applied voltage. The lines in ( b , c ) are exponential fits to the data. Experiments were performed in 0.5 M NaCl, 20 mM Tris, pH 7.5. Data shown in semi-log plots are mean and s.d. of 23,516 events (total for all points) from one hybrid nanopore

    Techniques Used:

    Design of a bio-inspired lipid-free hybrid nanopore. a Cartoon of the DNA packaging machine of a dsDNA virus. Viral genomic DNA (red) is translocated into the preformed virus capsid by the packaging ATPase (yellow) through the portal protein (aqua) embedded in viral capsid (gray). b Left, electrostatic properties of the tunnel in wild-type and mutant portal proteins. Slice through the middle of molecular surface colored according to charge from red (−1 kT e −1 ) to blue (+1 kT e −1 ). b Right (gray box), dimensions of the portal protein (teal, L) and the SS nanopore (pink, R). c Insertion of the purified portal protein into a nanopore fabricated in a thin solid-state (SS) membrane. Portal protein is applied to the trans chamber of a SS nanopore device containing an electrolyte solution of 20 mM Tris pH 7.5, 0.5 M NaCl. The protein electrokinetically inserts into the SS pore during application of a positive voltage. d Cartoon image of the hybrid pore, in which application of voltage results in ion current through the pore (blue arrows), as well as leakage current that is peripheral to the pore (red arrows)
    Figure Legend Snippet: Design of a bio-inspired lipid-free hybrid nanopore. a Cartoon of the DNA packaging machine of a dsDNA virus. Viral genomic DNA (red) is translocated into the preformed virus capsid by the packaging ATPase (yellow) through the portal protein (aqua) embedded in viral capsid (gray). b Left, electrostatic properties of the tunnel in wild-type and mutant portal proteins. Slice through the middle of molecular surface colored according to charge from red (−1 kT e −1 ) to blue (+1 kT e −1 ). b Right (gray box), dimensions of the portal protein (teal, L) and the SS nanopore (pink, R). c Insertion of the purified portal protein into a nanopore fabricated in a thin solid-state (SS) membrane. Portal protein is applied to the trans chamber of a SS nanopore device containing an electrolyte solution of 20 mM Tris pH 7.5, 0.5 M NaCl. The protein electrokinetically inserts into the SS pore during application of a positive voltage. d Cartoon image of the hybrid pore, in which application of voltage results in ion current through the pore (blue arrows), as well as leakage current that is peripheral to the pore (red arrows)

    Techniques Used: Mutagenesis, Purification

    Related Articles

    Irradiation:

    Article Title: Intrinsic Buffer Hydroxyl Radical Dosimetry Using Tris(Hydroxymethyl)Aminomethane
    Article Snippet: .. Upon comparing the UV absorbance spectra of Tris (obtained with a NanoDrop spectrometer) both with and without laser irradiation, we observed that, while Tris buffer is inherently absorbent in the short wavelength region of the ultraviolet spectrum, this absorbance changes little after oxidation by FPOP. ..

    Chromatography:

    Article Title: Plant cell wall glycosyltransferases: High-throughput recombinant expression screening and general requirements for these challenging enzymes
    Article Snippet: .. Briefly, 3 μg recombinant Arabidopsis thaliana RGP1 and 1 mM UDP-Araf were added to 20 mM Tris, 5 mM MnCl2 , 250 mM NaCl, pH 6.8, and incubated at 30°C for 1 h. The product was detected with high-performance anion-exchange chromatography using a Dionex Ultimate 3000 with CarboPac PA20 column (3 × 150 mm; Dionex; Sunnyvale, USA). .. Nucleotide sugars were separated with 0 to 2.1 min, 50 mM ammonium formate, isocratic; 2.1 to 40 min, 50 mM to 1 M ammonium formate, linear, 40 to 45 min, 1 M to 50 mM ammonium formate, linear, 45 to 55 min, 50 mM ammonium formate, isocratic, all at a flow rate of 0.5 mL/min.

    Protease Inhibitor:

    Article Title: The conformation of the histone H3 tail inhibits association of the BPTF PHD finger with the nucleosome
    Article Snippet: .. To purify H3(1–44) constructs, cells were lysed in 50 mM Tris pH 7.5, 100 mM NaCl, 1 mM benzamidine, 2 mM EDTA, 0.5% Triton X-100, 0.5 mg mL−1 lysozyme with DnaseI and Pierce Protease Inhibitor Tablets (ThermoFisher Scientific) using an Avestin EmulsiFlex. .. The soluble portion of the lysate was purified using cation exchange resin and size exclusion chromatography (Superdex 30, GE Healthcare Life Sciences) in 50 mM KPi pH 7, 50 mM KCl, 0.5 mM EDTA.

    Construct:

    Article Title: The conformation of the histone H3 tail inhibits association of the BPTF PHD finger with the nucleosome
    Article Snippet: .. To purify H3(1–44) constructs, cells were lysed in 50 mM Tris pH 7.5, 100 mM NaCl, 1 mM benzamidine, 2 mM EDTA, 0.5% Triton X-100, 0.5 mg mL−1 lysozyme with DnaseI and Pierce Protease Inhibitor Tablets (ThermoFisher Scientific) using an Avestin EmulsiFlex. .. The soluble portion of the lysate was purified using cation exchange resin and size exclusion chromatography (Superdex 30, GE Healthcare Life Sciences) in 50 mM KPi pH 7, 50 mM KCl, 0.5 mM EDTA.

    Purification:

    Article Title: Monodisperse 130 kDa and 260 kDa Recombinant Human Hemoglobin Polymers as Scaffolds for Protein Engineering of Hemoglobin-Based Oxygen Carriers
    Article Snippet: .. Tetrahemoglobin was produced from SGE2812 by treating the purified deoxygenated dihemoglobin at 150 mg/mL in 50 mM Tris pH 8.0 with a two-fold excess of bismaleimidohexane (Pierce, Inc., Salinas, CA, USA) for 2 h on ice. .. The crosslinked tetrahemoglobin was purified as described above using S-200 and S-300 SEC, followed by Q-Sepharose to remove endotoxins prior to formulation for preclinical testing.

    Article Title: Control of VWF A2 domain stability and ADAMTS13 access to the scissile bond of full-length VWF
    Article Snippet: .. Purified VWF A2 domain variants were dialyzed into 20 mM Tris (pH 7.8), 50 mM NaCl, and protein quantified by bicinchoninic acid assay (Pierce; Thermo Scientific). .. Samples contained 1.5 µg of purified VWF A2 domain, 5× SyproOrange dye (Sigma-Aldrich), 1 mM EDTA or 5mM CaCl2 in 20 mM Tris (pH 7.8), 50 mM NaCl in a total volume of 60 µL.

    Acid Assay:

    Article Title: Control of VWF A2 domain stability and ADAMTS13 access to the scissile bond of full-length VWF
    Article Snippet: .. Purified VWF A2 domain variants were dialyzed into 20 mM Tris (pH 7.8), 50 mM NaCl, and protein quantified by bicinchoninic acid assay (Pierce; Thermo Scientific). .. Samples contained 1.5 µg of purified VWF A2 domain, 5× SyproOrange dye (Sigma-Aldrich), 1 mM EDTA or 5mM CaCl2 in 20 mM Tris (pH 7.8), 50 mM NaCl in a total volume of 60 µL.

    Incubation:

    Article Title: Plant cell wall glycosyltransferases: High-throughput recombinant expression screening and general requirements for these challenging enzymes
    Article Snippet: .. Briefly, 3 μg recombinant Arabidopsis thaliana RGP1 and 1 mM UDP-Araf were added to 20 mM Tris, 5 mM MnCl2 , 250 mM NaCl, pH 6.8, and incubated at 30°C for 1 h. The product was detected with high-performance anion-exchange chromatography using a Dionex Ultimate 3000 with CarboPac PA20 column (3 × 150 mm; Dionex; Sunnyvale, USA). .. Nucleotide sugars were separated with 0 to 2.1 min, 50 mM ammonium formate, isocratic; 2.1 to 40 min, 50 mM to 1 M ammonium formate, linear, 40 to 45 min, 1 M to 50 mM ammonium formate, linear, 45 to 55 min, 50 mM ammonium formate, isocratic, all at a flow rate of 0.5 mL/min.

    Produced:

    Article Title: Monodisperse 130 kDa and 260 kDa Recombinant Human Hemoglobin Polymers as Scaffolds for Protein Engineering of Hemoglobin-Based Oxygen Carriers
    Article Snippet: .. Tetrahemoglobin was produced from SGE2812 by treating the purified deoxygenated dihemoglobin at 150 mg/mL in 50 mM Tris pH 8.0 with a two-fold excess of bismaleimidohexane (Pierce, Inc., Salinas, CA, USA) for 2 h on ice. .. The crosslinked tetrahemoglobin was purified as described above using S-200 and S-300 SEC, followed by Q-Sepharose to remove endotoxins prior to formulation for preclinical testing.

    Recombinant:

    Article Title: Plant cell wall glycosyltransferases: High-throughput recombinant expression screening and general requirements for these challenging enzymes
    Article Snippet: .. Briefly, 3 μg recombinant Arabidopsis thaliana RGP1 and 1 mM UDP-Araf were added to 20 mM Tris, 5 mM MnCl2 , 250 mM NaCl, pH 6.8, and incubated at 30°C for 1 h. The product was detected with high-performance anion-exchange chromatography using a Dionex Ultimate 3000 with CarboPac PA20 column (3 × 150 mm; Dionex; Sunnyvale, USA). .. Nucleotide sugars were separated with 0 to 2.1 min, 50 mM ammonium formate, isocratic; 2.1 to 40 min, 50 mM to 1 M ammonium formate, linear, 40 to 45 min, 1 M to 50 mM ammonium formate, linear, 45 to 55 min, 50 mM ammonium formate, isocratic, all at a flow rate of 0.5 mL/min.

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    Thermo Fisher pcx tri 2a
    Changes in Ikgkb ( Nemo ) mRNA expression in control (Ctrl) and <t>pCX-TRI-2A-transfected</t> cells. Cells were incubated with 50 ng/ml TNF-α for 16h, alone or in combination with 20 μM hemin and/or 200 μM ATP. Murine Ikgkb mRNA was quantified by real-time PCR. The data (mean±SD of three independent experiments), normalized for Gapdh gene, are expressed as fold change respect to the untreated cells. [*] indicates a significant difference between pCX-TRI-2A-transfected cells and Ctrl cells within the same treatment (t Student, p
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    Electrophoretic analysis of U 1 -SCRTX-Lg1a. Silver-stained 12% <t>SDS-PAGE</t> gel of the crude venom of L. gaucho (CV) (5 µg) and purified antimicrobial U 1 -SCRTX-Lg1a (2.5 µg) under non-reducing conditions. On the left are numbers that correspond to the positions of molecular weight markers (MW) expressed in kDa.
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    Thermo Fisher bis tris page sds gels
    Analysis of A244_N332-rgp120 secreted from stable MGAT1 - CHO cell lines. Six stable MGAT1 - CHO cell lines identified with the ClonePix2 were selected as potential substrates for HIV vaccine production. (A) Immunoblot of affinity-purified rgp120 (50 ng per lane) produced by each of six A244_N332-rgp120 cell lines: 3E, 5C, 5D, 3F, 6B, and 5F. Purified A244_N332-rgp120 produced in normal CHO DG44 cells (692) was shown for purpose of comparison. (B) Comparison of A244_N332-rgp120 protein yields as determined by ELISA from the six MGAT1 - CHO cell lines. ( C) <t>SDS</t> <t>PAGE</t> of rgp120 produced by the 5F MGAT1 - CHO cell line. Supernatant samples (10 μ l per lane) collected over the time course of the culture were electrophoresed on a 4–12% NuPage PAGE SDS gel in MOPS buffer (Thermo Scientific, Waltham, MA). The gel was stained with Simply Blue (Thermo Scientific, Waltham, MA) and visualized using an Innotech FluoChem2 system (Genetic Technologies, Grover, MO).
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    Thermo Fisher criterion tris triton precast sds page gels
    ADAM8 protein level correlates with PrP C1/full-length ratio in skeletal muscle tissue of mice constitutively expressing mouse PrP at different levels. Skeletal muscles (quadriceps) from the hind legs of wild type C57BL6 mice (BL6) and the Tga20 line constitutively expressing mouse PrP at high levels were collected. Muscle homogenates were either treated (for PrP probing) or not treated (for ADAM8 probing) with PNGase F, and subjected to <t>SDS-PAGE</t> and immunoblotting with an anti-PrP antibody (8H4) or an antibody against ADAM8. A , immunoblot probed with anti-PrP antibody 8H4 followed by probing with an anti-actin antibody after stripping. B , immunoblot probed with an antibody against ADAM8 followed by probing with an anti-actin antibody after stripping. C , plot of PrP C1/full-length ratios against relative ADAM8 protein levels. The error bars denote standard errors calculated from the three animals for each mouse line. The bars with an asterisk indicate statistical significance ( p
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    Changes in Ikgkb ( Nemo ) mRNA expression in control (Ctrl) and pCX-TRI-2A-transfected cells. Cells were incubated with 50 ng/ml TNF-α for 16h, alone or in combination with 20 μM hemin and/or 200 μM ATP. Murine Ikgkb mRNA was quantified by real-time PCR. The data (mean±SD of three independent experiments), normalized for Gapdh gene, are expressed as fold change respect to the untreated cells. [*] indicates a significant difference between pCX-TRI-2A-transfected cells and Ctrl cells within the same treatment (t Student, p

    Journal: PLoS ONE

    Article Title: Simultaneous Overexpression of Functional Human HO-1, E5NT and ENTPD1 Protects Murine Fibroblasts against TNF-α-Induced Injury In Vitro

    doi: 10.1371/journal.pone.0141933

    Figure Lengend Snippet: Changes in Ikgkb ( Nemo ) mRNA expression in control (Ctrl) and pCX-TRI-2A-transfected cells. Cells were incubated with 50 ng/ml TNF-α for 16h, alone or in combination with 20 μM hemin and/or 200 μM ATP. Murine Ikgkb mRNA was quantified by real-time PCR. The data (mean±SD of three independent experiments), normalized for Gapdh gene, are expressed as fold change respect to the untreated cells. [*] indicates a significant difference between pCX-TRI-2A-transfected cells and Ctrl cells within the same treatment (t Student, p

    Article Snippet: For Nfkb nuclear translocation analyses, pCX-TRI-2A and control cells were exposed to TNF-α 50ng/ml alone or in combination with hemin 20μM and ATP 200μM for 16h and proteins were extracted with NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific), following the manufacturer’s instructions.

    Techniques: Expressing, Transfection, Incubation, Real-time Polymerase Chain Reaction

    Changes in Tnfaip3 ( A20 ) mRNA expression in control (Ctrl) and pCX-TRI-2A-transfected cells. Cells were incubated with 50 ng/ml TNF-α for 16h, alone or in combination with 20 μM hemin and/or 200 μM ATP. Murine Tnfaip3/A20 mRNA was quantified by real-time PCR. Data (mean±SD of three independent experiments) are normalized for Gapdh gene and expressed as fold change respect to the untreated cells. [*] indicates a significant difference between pCX-TRI-2A-transfected cells and control cells within the same treatment (t Student, p

    Journal: PLoS ONE

    Article Title: Simultaneous Overexpression of Functional Human HO-1, E5NT and ENTPD1 Protects Murine Fibroblasts against TNF-α-Induced Injury In Vitro

    doi: 10.1371/journal.pone.0141933

    Figure Lengend Snippet: Changes in Tnfaip3 ( A20 ) mRNA expression in control (Ctrl) and pCX-TRI-2A-transfected cells. Cells were incubated with 50 ng/ml TNF-α for 16h, alone or in combination with 20 μM hemin and/or 200 μM ATP. Murine Tnfaip3/A20 mRNA was quantified by real-time PCR. Data (mean±SD of three independent experiments) are normalized for Gapdh gene and expressed as fold change respect to the untreated cells. [*] indicates a significant difference between pCX-TRI-2A-transfected cells and control cells within the same treatment (t Student, p

    Article Snippet: For Nfkb nuclear translocation analyses, pCX-TRI-2A and control cells were exposed to TNF-α 50ng/ml alone or in combination with hemin 20μM and ATP 200μM for 16h and proteins were extracted with NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific), following the manufacturer’s instructions.

    Techniques: Expressing, Transfection, Incubation, Real-time Polymerase Chain Reaction

    pCX TRI 2A-transfected cells were enriched via FACS for the expression of hE5NT and hENTPD1. Expression analysis on sorted transfectants showed that E5NT and ENTPD1 were expressed by more than 95% of the pCX-TRI-2A-transfected cells ( A ) and that more than 93% of those cells expressed both the two ecto-enzyme simultaneously ( B ). ( C ) The three human proteins were found strongly expressed into the transfected cells after sorting, and expression levels were unaffected by the number of genes in the construct, as there was no evidence of incomplete separation of individual proteins.

    Journal: PLoS ONE

    Article Title: Simultaneous Overexpression of Functional Human HO-1, E5NT and ENTPD1 Protects Murine Fibroblasts against TNF-α-Induced Injury In Vitro

    doi: 10.1371/journal.pone.0141933

    Figure Lengend Snippet: pCX TRI 2A-transfected cells were enriched via FACS for the expression of hE5NT and hENTPD1. Expression analysis on sorted transfectants showed that E5NT and ENTPD1 were expressed by more than 95% of the pCX-TRI-2A-transfected cells ( A ) and that more than 93% of those cells expressed both the two ecto-enzyme simultaneously ( B ). ( C ) The three human proteins were found strongly expressed into the transfected cells after sorting, and expression levels were unaffected by the number of genes in the construct, as there was no evidence of incomplete separation of individual proteins.

    Article Snippet: For Nfkb nuclear translocation analyses, pCX-TRI-2A and control cells were exposed to TNF-α 50ng/ml alone or in combination with hemin 20μM and ATP 200μM for 16h and proteins were extracted with NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific), following the manufacturer’s instructions.

    Techniques: Transfection, FACS, Expressing, Construct

    Nf-kB1 nuclear translocation in control (Ctrl) and pCX-TRI-2A-transfected cells. Cells were incubated with 50 ng/ml TNF-α for 16h, alone or in combination with 20 μM hemin and 200 μM ATP. Murine Nf-kB1/p50 protein was detected in nuclear extracts of Ctrl and pCX-TRI-2A-transfected cells by immunoblotting analysis ( A ). Densitometric analysis for Nf-kB1/p50, normalized for the Lamin B2 signal, showed a 60% or 83.6% increase in Nf-kB1/p50 level in pCX-TRI-2A-transfected cells treated with TNF-α alone or in combination with hemin and ATP, respectively, as compared to control cells ( B ). Densitometric analysis was performed by using “Gel analyzer” function of ImageJ software. Data are shown as mean ± S.D from at least three independent experiments. [*] indicates a significant difference between pCX-TRI-2A-transfected cells and control cells within the same treatment (t Student, p

    Journal: PLoS ONE

    Article Title: Simultaneous Overexpression of Functional Human HO-1, E5NT and ENTPD1 Protects Murine Fibroblasts against TNF-α-Induced Injury In Vitro

    doi: 10.1371/journal.pone.0141933

    Figure Lengend Snippet: Nf-kB1 nuclear translocation in control (Ctrl) and pCX-TRI-2A-transfected cells. Cells were incubated with 50 ng/ml TNF-α for 16h, alone or in combination with 20 μM hemin and 200 μM ATP. Murine Nf-kB1/p50 protein was detected in nuclear extracts of Ctrl and pCX-TRI-2A-transfected cells by immunoblotting analysis ( A ). Densitometric analysis for Nf-kB1/p50, normalized for the Lamin B2 signal, showed a 60% or 83.6% increase in Nf-kB1/p50 level in pCX-TRI-2A-transfected cells treated with TNF-α alone or in combination with hemin and ATP, respectively, as compared to control cells ( B ). Densitometric analysis was performed by using “Gel analyzer” function of ImageJ software. Data are shown as mean ± S.D from at least three independent experiments. [*] indicates a significant difference between pCX-TRI-2A-transfected cells and control cells within the same treatment (t Student, p

    Article Snippet: For Nfkb nuclear translocation analyses, pCX-TRI-2A and control cells were exposed to TNF-α 50ng/ml alone or in combination with hemin 20μM and ATP 200μM for 16h and proteins were extracted with NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific), following the manufacturer’s instructions.

    Techniques: Translocation Assay, Transfection, Incubation, Software

    Schematic maps of tricistronic pCX-TRI-2A vector and all the constructs used in the work. The transgenic plasmid is composed of the CAGGS promoter, followed by the coding sequence of human HO-1 gene without the stop codon, fused in frame to the first F2A sequence (F2A), then the coding sequence of human E5NT gene without the stop codon, the second F2A sequence and the coding sequence of human ENTPD1 gene followed by a polyadenilation signal (pA).

    Journal: PLoS ONE

    Article Title: Simultaneous Overexpression of Functional Human HO-1, E5NT and ENTPD1 Protects Murine Fibroblasts against TNF-α-Induced Injury In Vitro

    doi: 10.1371/journal.pone.0141933

    Figure Lengend Snippet: Schematic maps of tricistronic pCX-TRI-2A vector and all the constructs used in the work. The transgenic plasmid is composed of the CAGGS promoter, followed by the coding sequence of human HO-1 gene without the stop codon, fused in frame to the first F2A sequence (F2A), then the coding sequence of human E5NT gene without the stop codon, the second F2A sequence and the coding sequence of human ENTPD1 gene followed by a polyadenilation signal (pA).

    Article Snippet: For Nfkb nuclear translocation analyses, pCX-TRI-2A and control cells were exposed to TNF-α 50ng/ml alone or in combination with hemin 20μM and ATP 200μM for 16h and proteins were extracted with NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific), following the manufacturer’s instructions.

    Techniques: Plasmid Preparation, Construct, Transgenic Assay, Sequencing

    pCX-TRI-2A transfected cells are protected against TNF-α-induced cytotoxicity. WT cells and transfected cell lines were incubated with 50 ng/ml TNF-α for 24 h ( A ) and 48 h ( B ) alone or in combination with 20 μM hemin and/or 200 μM ATP. Cytotoxicity was assessed by lactate dehydrogenase (LDH) release and expressed as follows: relative cytotoxicity (%) = [( A e − A c) / ( A b − A c)] × 100 (%), where ‘ A e’ is the experimental absorbance, ‘ A b’ is the absorbance of lysed controls and ‘ A c’ is the absorbance of untreated controls. The data are expressed as mean ± SD of three independent experiments. [#] indicates significant difference between pCX-TRI-2A and all the other groups, except for pCX-hHO1, within the same treatment (ANOVA, p

    Journal: PLoS ONE

    Article Title: Simultaneous Overexpression of Functional Human HO-1, E5NT and ENTPD1 Protects Murine Fibroblasts against TNF-α-Induced Injury In Vitro

    doi: 10.1371/journal.pone.0141933

    Figure Lengend Snippet: pCX-TRI-2A transfected cells are protected against TNF-α-induced cytotoxicity. WT cells and transfected cell lines were incubated with 50 ng/ml TNF-α for 24 h ( A ) and 48 h ( B ) alone or in combination with 20 μM hemin and/or 200 μM ATP. Cytotoxicity was assessed by lactate dehydrogenase (LDH) release and expressed as follows: relative cytotoxicity (%) = [( A e − A c) / ( A b − A c)] × 100 (%), where ‘ A e’ is the experimental absorbance, ‘ A b’ is the absorbance of lysed controls and ‘ A c’ is the absorbance of untreated controls. The data are expressed as mean ± SD of three independent experiments. [#] indicates significant difference between pCX-TRI-2A and all the other groups, except for pCX-hHO1, within the same treatment (ANOVA, p

    Article Snippet: For Nfkb nuclear translocation analyses, pCX-TRI-2A and control cells were exposed to TNF-α 50ng/ml alone or in combination with hemin 20μM and ATP 200μM for 16h and proteins were extracted with NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific), following the manufacturer’s instructions.

    Techniques: Transfection, Incubation

    All the three exogenous proteins were correctly localized in pCX TRI 2A-transfected cells. WT and pCX-TRI-2A-transfected cells were co-stained with anti-hHO1 and anti-hE5NT antibodies ( A ) or with anti-hHO1and anti-hENTPD1antibodies ( B ). Transfected cells positive to hE5NT or hENTPD1 (red) were also positive to hHO1 (green). hE5NT and hENTPD1 localized on the cell surface, while hHO1 had a perinuclear and/or ER membranes cytoplasmic localization. ( C - D ) Plot of the signal intensity for hE5NT and hENTPD1 (red) and hHO1 (green) along the line drawn in A and B indicated that the most intense hE5NT and hENTPD1 signals are not colocalized with hHO1 signal.

    Journal: PLoS ONE

    Article Title: Simultaneous Overexpression of Functional Human HO-1, E5NT and ENTPD1 Protects Murine Fibroblasts against TNF-α-Induced Injury In Vitro

    doi: 10.1371/journal.pone.0141933

    Figure Lengend Snippet: All the three exogenous proteins were correctly localized in pCX TRI 2A-transfected cells. WT and pCX-TRI-2A-transfected cells were co-stained with anti-hHO1 and anti-hE5NT antibodies ( A ) or with anti-hHO1and anti-hENTPD1antibodies ( B ). Transfected cells positive to hE5NT or hENTPD1 (red) were also positive to hHO1 (green). hE5NT and hENTPD1 localized on the cell surface, while hHO1 had a perinuclear and/or ER membranes cytoplasmic localization. ( C - D ) Plot of the signal intensity for hE5NT and hENTPD1 (red) and hHO1 (green) along the line drawn in A and B indicated that the most intense hE5NT and hENTPD1 signals are not colocalized with hHO1 signal.

    Article Snippet: For Nfkb nuclear translocation analyses, pCX-TRI-2A and control cells were exposed to TNF-α 50ng/ml alone or in combination with hemin 20μM and ATP 200μM for 16h and proteins were extracted with NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific), following the manufacturer’s instructions.

    Techniques: Transfection, Staining

    pCX-TRI-2A transfected-cells are protected against TNF-α-induced apoptosis. Caspase 3/7 activities were determined in all cell groups after 16h ( A ) or 24h ( B ) of incubation with 50 ng/ml TNF-α alone or in combination with 20 μM hemin and/or 200 μM ATP. Expression of the three human genes in TG cells significantly reduced the activation of effector caspases 3/7. The data are expressed as mean ± SD of three independent experiments. [#] indicates significant difference between pCX-TRI-2A and all the other groups, except for pCX-hHO1, within the same treatment (ANOVA, p

    Journal: PLoS ONE

    Article Title: Simultaneous Overexpression of Functional Human HO-1, E5NT and ENTPD1 Protects Murine Fibroblasts against TNF-α-Induced Injury In Vitro

    doi: 10.1371/journal.pone.0141933

    Figure Lengend Snippet: pCX-TRI-2A transfected-cells are protected against TNF-α-induced apoptosis. Caspase 3/7 activities were determined in all cell groups after 16h ( A ) or 24h ( B ) of incubation with 50 ng/ml TNF-α alone or in combination with 20 μM hemin and/or 200 μM ATP. Expression of the three human genes in TG cells significantly reduced the activation of effector caspases 3/7. The data are expressed as mean ± SD of three independent experiments. [#] indicates significant difference between pCX-TRI-2A and all the other groups, except for pCX-hHO1, within the same treatment (ANOVA, p

    Article Snippet: For Nfkb nuclear translocation analyses, pCX-TRI-2A and control cells were exposed to TNF-α 50ng/ml alone or in combination with hemin 20μM and ATP 200μM for 16h and proteins were extracted with NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific), following the manufacturer’s instructions.

    Techniques: Transfection, Incubation, Expressing, Activation Assay

    Heme oxygenase-1 and ectonucleotidases functional assays. ( A ) Heme Oxygenase 1 activity assay on NIH3T3 cells. Lysates from WT, mock- and pCX-TRI-2A-transfected cells were incubated for 2 hours with Hemin, BSA, Biliverdin Reductase A in reaction buffer as described in Materials and Methods. As positive control of the assay, wt or mock-transfected cells were pre-stimulated with 50 μM of HO1 inducer, Cobalt Protoporphyrin for 24 hours (wt CoPP, mock CoPP). Enzymatic activity is reported as nanomoles of bilirubin per hours per milligrams of protein extract. Data are expressed as mean ± SEM of 3–4 independent experiments. * p

    Journal: PLoS ONE

    Article Title: Simultaneous Overexpression of Functional Human HO-1, E5NT and ENTPD1 Protects Murine Fibroblasts against TNF-α-Induced Injury In Vitro

    doi: 10.1371/journal.pone.0141933

    Figure Lengend Snippet: Heme oxygenase-1 and ectonucleotidases functional assays. ( A ) Heme Oxygenase 1 activity assay on NIH3T3 cells. Lysates from WT, mock- and pCX-TRI-2A-transfected cells were incubated for 2 hours with Hemin, BSA, Biliverdin Reductase A in reaction buffer as described in Materials and Methods. As positive control of the assay, wt or mock-transfected cells were pre-stimulated with 50 μM of HO1 inducer, Cobalt Protoporphyrin for 24 hours (wt CoPP, mock CoPP). Enzymatic activity is reported as nanomoles of bilirubin per hours per milligrams of protein extract. Data are expressed as mean ± SEM of 3–4 independent experiments. * p

    Article Snippet: For Nfkb nuclear translocation analyses, pCX-TRI-2A and control cells were exposed to TNF-α 50ng/ml alone or in combination with hemin 20μM and ATP 200μM for 16h and proteins were extracted with NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific), following the manufacturer’s instructions.

    Techniques: Functional Assay, Activity Assay, Transfection, Incubation, Positive Control

    Electrophoretic analysis of U 1 -SCRTX-Lg1a. Silver-stained 12% SDS-PAGE gel of the crude venom of L. gaucho (CV) (5 µg) and purified antimicrobial U 1 -SCRTX-Lg1a (2.5 µg) under non-reducing conditions. On the left are numbers that correspond to the positions of molecular weight markers (MW) expressed in kDa.

    Journal: Toxins

    Article Title: Loxosceles gaucho Spider Venom: An Untapped Source of Antimicrobial Agents

    doi: 10.3390/toxins10120522

    Figure Lengend Snippet: Electrophoretic analysis of U 1 -SCRTX-Lg1a. Silver-stained 12% SDS-PAGE gel of the crude venom of L. gaucho (CV) (5 µg) and purified antimicrobial U 1 -SCRTX-Lg1a (2.5 µg) under non-reducing conditions. On the left are numbers that correspond to the positions of molecular weight markers (MW) expressed in kDa.

    Article Snippet: SDS-PAGE Analysis Samples of L. gaucho crude venom (5 µg) and U1 -SCRTX-Lg1a (2.5 µg) were analyzed by 12% tris-glycine SDS-PAGE under non-reducing conditions [ ], using a Spectra Multicolor Broad Range Protein Ladder (Thermo Fisher Scientific, Waltham, MA, USA) to estimate the molecular mass.

    Techniques: Staining, SDS Page, Purification, Molecular Weight

    Analysis of A244_N332-rgp120 secreted from stable MGAT1 - CHO cell lines. Six stable MGAT1 - CHO cell lines identified with the ClonePix2 were selected as potential substrates for HIV vaccine production. (A) Immunoblot of affinity-purified rgp120 (50 ng per lane) produced by each of six A244_N332-rgp120 cell lines: 3E, 5C, 5D, 3F, 6B, and 5F. Purified A244_N332-rgp120 produced in normal CHO DG44 cells (692) was shown for purpose of comparison. (B) Comparison of A244_N332-rgp120 protein yields as determined by ELISA from the six MGAT1 - CHO cell lines. ( C) SDS PAGE of rgp120 produced by the 5F MGAT1 - CHO cell line. Supernatant samples (10 μ l per lane) collected over the time course of the culture were electrophoresed on a 4–12% NuPage PAGE SDS gel in MOPS buffer (Thermo Scientific, Waltham, MA). The gel was stained with Simply Blue (Thermo Scientific, Waltham, MA) and visualized using an Innotech FluoChem2 system (Genetic Technologies, Grover, MO).

    Journal: PLoS ONE

    Article Title: Robotic selection for the rapid development of stable CHO cell lines for HIV vaccine production

    doi: 10.1371/journal.pone.0197656

    Figure Lengend Snippet: Analysis of A244_N332-rgp120 secreted from stable MGAT1 - CHO cell lines. Six stable MGAT1 - CHO cell lines identified with the ClonePix2 were selected as potential substrates for HIV vaccine production. (A) Immunoblot of affinity-purified rgp120 (50 ng per lane) produced by each of six A244_N332-rgp120 cell lines: 3E, 5C, 5D, 3F, 6B, and 5F. Purified A244_N332-rgp120 produced in normal CHO DG44 cells (692) was shown for purpose of comparison. (B) Comparison of A244_N332-rgp120 protein yields as determined by ELISA from the six MGAT1 - CHO cell lines. ( C) SDS PAGE of rgp120 produced by the 5F MGAT1 - CHO cell line. Supernatant samples (10 μ l per lane) collected over the time course of the culture were electrophoresed on a 4–12% NuPage PAGE SDS gel in MOPS buffer (Thermo Scientific, Waltham, MA). The gel was stained with Simply Blue (Thermo Scientific, Waltham, MA) and visualized using an Innotech FluoChem2 system (Genetic Technologies, Grover, MO).

    Article Snippet: Purified protein and cell culture supernatant were analyzed on 4–12% Bis-Tris PAGE SDS gels in either MES or MOPS gel running buffer (Thermo Scientific, Waltham, MA).

    Techniques: Affinity Purification, Produced, Purification, Enzyme-linked Immunosorbent Assay, SDS Page, Polyacrylamide Gel Electrophoresis, SDS-Gel, Staining

    SDS-PAGE analysis of A244_N332 rgp120 HIV produced in 5F MGAT1 - CHO and CHO-S cells treated with PNGase or EndoH. Enzymes and buffers were purchased from (New England Biolabs, Ipswich, MA). Purified protein was denatured and reduced then incubated overnight at 37°C with or without glycosidase. Protein was resolved (2 μg/lane) on 4–12% SDS-PAGE gel and stained with Simply Blue. Plus (+) indicates enzyme treatment, minus indicates untreated.

    Journal: PLoS ONE

    Article Title: Robotic selection for the rapid development of stable CHO cell lines for HIV vaccine production

    doi: 10.1371/journal.pone.0197656

    Figure Lengend Snippet: SDS-PAGE analysis of A244_N332 rgp120 HIV produced in 5F MGAT1 - CHO and CHO-S cells treated with PNGase or EndoH. Enzymes and buffers were purchased from (New England Biolabs, Ipswich, MA). Purified protein was denatured and reduced then incubated overnight at 37°C with or without glycosidase. Protein was resolved (2 μg/lane) on 4–12% SDS-PAGE gel and stained with Simply Blue. Plus (+) indicates enzyme treatment, minus indicates untreated.

    Article Snippet: Purified protein and cell culture supernatant were analyzed on 4–12% Bis-Tris PAGE SDS gels in either MES or MOPS gel running buffer (Thermo Scientific, Waltham, MA).

    Techniques: SDS Page, Produced, Purification, Incubation, Staining

    ADAM8 protein level correlates with PrP C1/full-length ratio in skeletal muscle tissue of mice constitutively expressing mouse PrP at different levels. Skeletal muscles (quadriceps) from the hind legs of wild type C57BL6 mice (BL6) and the Tga20 line constitutively expressing mouse PrP at high levels were collected. Muscle homogenates were either treated (for PrP probing) or not treated (for ADAM8 probing) with PNGase F, and subjected to SDS-PAGE and immunoblotting with an anti-PrP antibody (8H4) or an antibody against ADAM8. A , immunoblot probed with anti-PrP antibody 8H4 followed by probing with an anti-actin antibody after stripping. B , immunoblot probed with an antibody against ADAM8 followed by probing with an anti-actin antibody after stripping. C , plot of PrP C1/full-length ratios against relative ADAM8 protein levels. The error bars denote standard errors calculated from the three animals for each mouse line. The bars with an asterisk indicate statistical significance ( p

    Journal: The Journal of Biological Chemistry

    Article Title: Cellular Prion Protein Regulates Its Own ?-Cleavage through ADAM8 in Skeletal Muscle *

    doi: 10.1074/jbc.M112.360891

    Figure Lengend Snippet: ADAM8 protein level correlates with PrP C1/full-length ratio in skeletal muscle tissue of mice constitutively expressing mouse PrP at different levels. Skeletal muscles (quadriceps) from the hind legs of wild type C57BL6 mice (BL6) and the Tga20 line constitutively expressing mouse PrP at high levels were collected. Muscle homogenates were either treated (for PrP probing) or not treated (for ADAM8 probing) with PNGase F, and subjected to SDS-PAGE and immunoblotting with an anti-PrP antibody (8H4) or an antibody against ADAM8. A , immunoblot probed with anti-PrP antibody 8H4 followed by probing with an anti-actin antibody after stripping. B , immunoblot probed with an antibody against ADAM8 followed by probing with an anti-actin antibody after stripping. C , plot of PrP C1/full-length ratios against relative ADAM8 protein levels. The error bars denote standard errors calculated from the three animals for each mouse line. The bars with an asterisk indicate statistical significance ( p

    Article Snippet: The rest of the human PrP cleavage products were separated on 10–20% Criterion Tris-Triton Precast SDS-PAGE gels for Coomassie Blue staining with Imperial Protein Stain (Thermo Scientific, Rockford, IL).

    Techniques: Mouse Assay, Expressing, SDS Page, Stripping Membranes

    The PrP C1/full-length fragment ratio is significantly reduced in skeletal muscles of ADAM8 knock-out mice. Skeletal muscles (quadriceps) from the hind legs of three each of wild type C57BL/6 mice (Wt) and ADAM8 −/− mice were collected. Muscle homogenates were either treated (for PrP probing) or not treated (for ADAM8 probing) with PNGase F, subjected to SDS-PAGE, and probed with an anti-PrP antibody (8H4) or an antibody against ADAM8. A , immunoblots probed with an antibody against ADAM8 or 8H4 followed by probing with an anti-actin antibody after stripping. B , diagram of PrP C1/full-length ratio in the skeletal muscles of Wt and ADAM8 −/− mice. The error bars denote standard errors calculated from the three animals for each mouse line. The bars with asterisk (s) indicate statistical significance when compared with the Wt mouse samples. **, p

    Journal: The Journal of Biological Chemistry

    Article Title: Cellular Prion Protein Regulates Its Own ?-Cleavage through ADAM8 in Skeletal Muscle *

    doi: 10.1074/jbc.M112.360891

    Figure Lengend Snippet: The PrP C1/full-length fragment ratio is significantly reduced in skeletal muscles of ADAM8 knock-out mice. Skeletal muscles (quadriceps) from the hind legs of three each of wild type C57BL/6 mice (Wt) and ADAM8 −/− mice were collected. Muscle homogenates were either treated (for PrP probing) or not treated (for ADAM8 probing) with PNGase F, subjected to SDS-PAGE, and probed with an anti-PrP antibody (8H4) or an antibody against ADAM8. A , immunoblots probed with an antibody against ADAM8 or 8H4 followed by probing with an anti-actin antibody after stripping. B , diagram of PrP C1/full-length ratio in the skeletal muscles of Wt and ADAM8 −/− mice. The error bars denote standard errors calculated from the three animals for each mouse line. The bars with asterisk (s) indicate statistical significance when compared with the Wt mouse samples. **, p

    Article Snippet: The rest of the human PrP cleavage products were separated on 10–20% Criterion Tris-Triton Precast SDS-PAGE gels for Coomassie Blue staining with Imperial Protein Stain (Thermo Scientific, Rockford, IL).

    Techniques: Knock-Out, Mouse Assay, SDS Page, Western Blot, Stripping Membranes

    ADAM8 protein level correlates with PrP C1/full-length ratio in skeletal muscle tissue of Dox-induced Tg(HQK) mice. Tg(HQK) mice were treated with 6 g of Dox/kg food for 0–60 days as indicated and 3–6 animals were taken at each time point. Skeletal muscles (quadriceps) from the hind legs of these animals were homogenized and subjected to SDS-PAGE and immunoblot analysis in three Western blots. The homogenates were either treated (for PrP probing) or not treated (for ADAM8 probing) with PNGase F before SDS-PAGE. A , representative immunoblot probed with anti-PrP antibody 8H4 followed by probing with an anti-actin antibody after stripping; samples for day 3, 5, and 6 are not shown. B , representative immunoblot probed with an antibody against ADAM8 followed by probing with an anti-actin antibody after stripping; samples for day 3, 5, and 6 are not shown. C and D , diagrams of PrP C1/full-length ratios ( C ) and ADAM8 protein level ( D ) over increasing duration of Dox treatment. The ADAM8 protein level for each sample was normalized against the actin level for the sample in each blot and expressed as the ratio against the normalized ADAM8 protein level in the untreated wild type FVB mouse on the same blot. The error bars denote standard deviations calculated from the duplicate blots. E , plot of PrP C1/full-length ratio against ADAM8 protein level over the time course of Dox treatment.

    Journal: The Journal of Biological Chemistry

    Article Title: Cellular Prion Protein Regulates Its Own ?-Cleavage through ADAM8 in Skeletal Muscle *

    doi: 10.1074/jbc.M112.360891

    Figure Lengend Snippet: ADAM8 protein level correlates with PrP C1/full-length ratio in skeletal muscle tissue of Dox-induced Tg(HQK) mice. Tg(HQK) mice were treated with 6 g of Dox/kg food for 0–60 days as indicated and 3–6 animals were taken at each time point. Skeletal muscles (quadriceps) from the hind legs of these animals were homogenized and subjected to SDS-PAGE and immunoblot analysis in three Western blots. The homogenates were either treated (for PrP probing) or not treated (for ADAM8 probing) with PNGase F before SDS-PAGE. A , representative immunoblot probed with anti-PrP antibody 8H4 followed by probing with an anti-actin antibody after stripping; samples for day 3, 5, and 6 are not shown. B , representative immunoblot probed with an antibody against ADAM8 followed by probing with an anti-actin antibody after stripping; samples for day 3, 5, and 6 are not shown. C and D , diagrams of PrP C1/full-length ratios ( C ) and ADAM8 protein level ( D ) over increasing duration of Dox treatment. The ADAM8 protein level for each sample was normalized against the actin level for the sample in each blot and expressed as the ratio against the normalized ADAM8 protein level in the untreated wild type FVB mouse on the same blot. The error bars denote standard deviations calculated from the duplicate blots. E , plot of PrP C1/full-length ratio against ADAM8 protein level over the time course of Dox treatment.

    Article Snippet: The rest of the human PrP cleavage products were separated on 10–20% Criterion Tris-Triton Precast SDS-PAGE gels for Coomassie Blue staining with Imperial Protein Stain (Thermo Scientific, Rockford, IL).

    Techniques: Mouse Assay, SDS Page, Western Blot, Stripping Membranes

    ADAM8 directly cleaves PrP to generate the C1 fragment. Recombinant human PrP (rhPrP) was cleaved at 37 °C for 14 h with a recombinant human ADAM8 that had been treated or untreated with thermolysin for activation, followed by SDS-PAGE and subsequent immunoblot analysis ( A and B ) or direct staining with Coomassie Blue ( C ), or by direct mass spectrometry analysis ( D ). A , Western blot probed with antibodies 8H4 (against human PrP176–186). B , Western blot probed with 3F4 (against human PrP 106–112). C , SDS-PAGE gel directly stained with Coomassie Blue. D , mass spectrometry analysis. An in vitro reaction sample with thermolysin-activated ADAM8 and rhPrP was analyzed. Monoisotopic distributions of protonated (M+11H) 11+ N1 fragment ion ( top ) and protonated (M+13H) 13+ C1 fragment ion ( bottom ) are shown; Mm : molecular mass. In panels A-B , skeletal muscle tissue homogenate from a Tg(HQK) mouse treated with Dox for 60 days was loaded as a control. In panels A–C , the arrows point to the C1 band. The rhPrP sample contained an extension of Gly-Ser at the N terminus as a leftover from the His tag; accordingly, the resulting N1 fragment also contained the extra Gly-Ser residues at the N terminus.

    Journal: The Journal of Biological Chemistry

    Article Title: Cellular Prion Protein Regulates Its Own ?-Cleavage through ADAM8 in Skeletal Muscle *

    doi: 10.1074/jbc.M112.360891

    Figure Lengend Snippet: ADAM8 directly cleaves PrP to generate the C1 fragment. Recombinant human PrP (rhPrP) was cleaved at 37 °C for 14 h with a recombinant human ADAM8 that had been treated or untreated with thermolysin for activation, followed by SDS-PAGE and subsequent immunoblot analysis ( A and B ) or direct staining with Coomassie Blue ( C ), or by direct mass spectrometry analysis ( D ). A , Western blot probed with antibodies 8H4 (against human PrP176–186). B , Western blot probed with 3F4 (against human PrP 106–112). C , SDS-PAGE gel directly stained with Coomassie Blue. D , mass spectrometry analysis. An in vitro reaction sample with thermolysin-activated ADAM8 and rhPrP was analyzed. Monoisotopic distributions of protonated (M+11H) 11+ N1 fragment ion ( top ) and protonated (M+13H) 13+ C1 fragment ion ( bottom ) are shown; Mm : molecular mass. In panels A-B , skeletal muscle tissue homogenate from a Tg(HQK) mouse treated with Dox for 60 days was loaded as a control. In panels A–C , the arrows point to the C1 band. The rhPrP sample contained an extension of Gly-Ser at the N terminus as a leftover from the His tag; accordingly, the resulting N1 fragment also contained the extra Gly-Ser residues at the N terminus.

    Article Snippet: The rest of the human PrP cleavage products were separated on 10–20% Criterion Tris-Triton Precast SDS-PAGE gels for Coomassie Blue staining with Imperial Protein Stain (Thermo Scientific, Rockford, IL).

    Techniques: Recombinant, Activation Assay, SDS Page, Staining, Mass Spectrometry, Western Blot, In Vitro

    ADAM8 protein level correlates with PrP C1/full-length ratio in skeletal muscle tissue of Tg mice constitutively expressing human PrP at different levels. Skeletal muscles (quadriceps) from the hind legs of wild type FVB mice (Wt) and four different Tg mice lines (Tg43, Tg4, Tg17, and Tg21) constitutively expressing human PrP at different levels were collected. Muscle homogenates were either treated (for PrP probing) or not treated (for ADAM8 probing) with PNGase F, subjected to SDS-PAGE, and probed with an anti-PrP antibody (8H4) or an antibody against ADAM8. A , representative immunoblot probed with anti-PrP antibody 8H4 followed by probing with an anti-actin antibody after stripping. B , representative immunoblot probed with an antibody against ADAM8 followed by probing with an anti-actin antibody after stripping. C and D , diagrams of ADAM8 protein level ( C ) and PrP C1/full-length ratios ( D ) in the skeletal muscles of the four Tg mouse lines. The ADAM8 protein level for each sample was normalized against the actin level and expressed as the ratio against the normalized ADAM8 protein level in the untreated sex and age-matched wild type FVB mouse on the same blot. The error bars denote standard errors calculated from the three animals for each Tg line. The bars with asterisk(s) indicate statistical significance when compared with the Tg43 mouse samples; *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Cellular Prion Protein Regulates Its Own ?-Cleavage through ADAM8 in Skeletal Muscle *

    doi: 10.1074/jbc.M112.360891

    Figure Lengend Snippet: ADAM8 protein level correlates with PrP C1/full-length ratio in skeletal muscle tissue of Tg mice constitutively expressing human PrP at different levels. Skeletal muscles (quadriceps) from the hind legs of wild type FVB mice (Wt) and four different Tg mice lines (Tg43, Tg4, Tg17, and Tg21) constitutively expressing human PrP at different levels were collected. Muscle homogenates were either treated (for PrP probing) or not treated (for ADAM8 probing) with PNGase F, subjected to SDS-PAGE, and probed with an anti-PrP antibody (8H4) or an antibody against ADAM8. A , representative immunoblot probed with anti-PrP antibody 8H4 followed by probing with an anti-actin antibody after stripping. B , representative immunoblot probed with an antibody against ADAM8 followed by probing with an anti-actin antibody after stripping. C and D , diagrams of ADAM8 protein level ( C ) and PrP C1/full-length ratios ( D ) in the skeletal muscles of the four Tg mouse lines. The ADAM8 protein level for each sample was normalized against the actin level and expressed as the ratio against the normalized ADAM8 protein level in the untreated sex and age-matched wild type FVB mouse on the same blot. The error bars denote standard errors calculated from the three animals for each Tg line. The bars with asterisk(s) indicate statistical significance when compared with the Tg43 mouse samples; *, p

    Article Snippet: The rest of the human PrP cleavage products were separated on 10–20% Criterion Tris-Triton Precast SDS-PAGE gels for Coomassie Blue staining with Imperial Protein Stain (Thermo Scientific, Rockford, IL).

    Techniques: Mouse Assay, Expressing, SDS Page, Stripping Membranes