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    Stratagene pcdna3 ns5a y93h
    Co-precipitation of BMS-Biotin with <t>NS5A</t> and <t>NS5A-Y93H.</t> (A) Genotype 1b, Con1 replicon cells (wild type or harboring the Y93H mutant of NS5A) were treated with 1μM DCV or 25 μM BMS-Biotin and incubated for 24 hr. Cells were harvested and the membrane fraction was solubilized with C12E8. Solubilized proteins were incubated with streptavidin agarose beads and co-precipitated material was subjected to SDS-PAGE. NS5A was visualized by immunoblot analysis using an anti-NS5A antibody. (B) Huh7-lunet cells were transfected with <t>pcDNA3-NS5A</t> or pcDNA3-NS5A-Y93H. 24 hr post transfection, cells were treated with the indicated amount of BMS-Biotin, incubated for 42 hr and processed as in (A).
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    Co-precipitation of BMS-Biotin with NS5A and NS5A-Y93H. (A) Genotype 1b, Con1 replicon cells (wild type or harboring the Y93H mutant of NS5A) were treated with 1μM DCV or 25 μM BMS-Biotin and incubated for 24 hr. Cells were harvested and the membrane fraction was solubilized with C12E8. Solubilized proteins were incubated with streptavidin agarose beads and co-precipitated material was subjected to SDS-PAGE. NS5A was visualized by immunoblot analysis using an anti-NS5A antibody. (B) Huh7-lunet cells were transfected with pcDNA3-NS5A or pcDNA3-NS5A-Y93H. 24 hr post transfection, cells were treated with the indicated amount of BMS-Biotin, incubated for 42 hr and processed as in (A).

    Journal: PLoS ONE

    Article Title: Direct Binding of Ledipasvir to HCV NS5A: Mechanism of Resistance to an HCV Antiviral Agent

    doi: 10.1371/journal.pone.0122844

    Figure Lengend Snippet: Co-precipitation of BMS-Biotin with NS5A and NS5A-Y93H. (A) Genotype 1b, Con1 replicon cells (wild type or harboring the Y93H mutant of NS5A) were treated with 1μM DCV or 25 μM BMS-Biotin and incubated for 24 hr. Cells were harvested and the membrane fraction was solubilized with C12E8. Solubilized proteins were incubated with streptavidin agarose beads and co-precipitated material was subjected to SDS-PAGE. NS5A was visualized by immunoblot analysis using an anti-NS5A antibody. (B) Huh7-lunet cells were transfected with pcDNA3-NS5A or pcDNA3-NS5A-Y93H. 24 hr post transfection, cells were treated with the indicated amount of BMS-Biotin, incubated for 42 hr and processed as in (A).

    Article Snippet: The PCR fragment was digested with Xba1 and EcoRV and ligated into pcDNA3.1(-) (Invitrogen). pcDNA3-NS5A-Y93H was generated by site-directed mutagenesis (Stratagene). pTK-NS5A was constructed by digesting pcDNA3-NS5A with Xba1 and EcoRV to isolate a 1.5 kb fragment encoding NS5A.

    Techniques: Mutagenesis, Incubation, SDS Page, Transfection

    3 H-LDV binding to NS5A-6HIS. (A) Each reaction, in a final volume of 200 μl, contained 50 nM of purified NS5A-6HIS and the indicated concentration of 3 H-LDV in the absence ( ) or presence (○) of unlabeled LDV. Bound 3 H-LDV was measured as described in Materials and Methods. Each data point represents the average of 4–7 assays. (B) Specific binding of 3 H-LDV to NS5A-6HIS (●) vs. NS5A-Y93H-6HIS ( ). Specific binding was defined as the difference between the amount of 3 H-LDV bound in the absence (total binding) and presence (non-specific binding) of unlabeled LDV. Each data point represents the average of at least 3 assays.

    Journal: PLoS ONE

    Article Title: Direct Binding of Ledipasvir to HCV NS5A: Mechanism of Resistance to an HCV Antiviral Agent

    doi: 10.1371/journal.pone.0122844

    Figure Lengend Snippet: 3 H-LDV binding to NS5A-6HIS. (A) Each reaction, in a final volume of 200 μl, contained 50 nM of purified NS5A-6HIS and the indicated concentration of 3 H-LDV in the absence ( ) or presence (○) of unlabeled LDV. Bound 3 H-LDV was measured as described in Materials and Methods. Each data point represents the average of 4–7 assays. (B) Specific binding of 3 H-LDV to NS5A-6HIS (●) vs. NS5A-Y93H-6HIS ( ). Specific binding was defined as the difference between the amount of 3 H-LDV bound in the absence (total binding) and presence (non-specific binding) of unlabeled LDV. Each data point represents the average of at least 3 assays.

    Article Snippet: The PCR fragment was digested with Xba1 and EcoRV and ligated into pcDNA3.1(-) (Invitrogen). pcDNA3-NS5A-Y93H was generated by site-directed mutagenesis (Stratagene). pTK-NS5A was constructed by digesting pcDNA3-NS5A with Xba1 and EcoRV to isolate a 1.5 kb fragment encoding NS5A.

    Techniques: Binding Assay, Purification, Concentration Assay

    Characterization of purified NS5A. (A) Coomassie staining of purified proteins. Recombinant His-tagged proteins were purified as described in Materials and Methods. 1 μg of NS5A-6HIS and NS5A-Y93H-6HIS were subjected to SDS-PAGE and the protein bands were visualized by Coomassie Blue stain. Molecular weights for protein standards are indicated. (B) Phosphorylation sites determined by mass spectrometry of purified NS5A-6HIS and NS5A-Y93H-6HIS. Phosphorylation sites identified in NS5A-6HIS are shaded green; sites identified in NS5A-Y93H-6HIS are shaded red; and sites identified in both proteins are shaded cyan. (C) Analytical ultracentrifugation analysis of NS5A-6HIS, NS5A-Y93H-6HIS, and NS5A-domain 1. For NS5A-6HIS and NS5A-Y93H-6HIS, analytical ultracentrifugation was performed in 25 mM Tris pH 7.5, 150 mM NaCl, 0.01% NaN 3 , 0.5 mM TCEP, 2 μg/ml Leupeptin, 0.02% C12E8, and 21.1% (v/v) D 2 O. Sedimentation velocity analysis was performed at 42,000 rpm for two protein concentrations (3.3 and 6.6 μM). For NS5A-domain 1, analytical ultracentrifugation was performed in 25 mM Tris pH 8.0, 250 mM NaCl, 10% glycerol, and 0.5% DMSO. Sedimentation velocity analysis was performed at 48,000 rpm for 4 protein concentrations (7.5, 12, 15, 30 μM). Representative traces are shown for each protein. (D) Circular dichroism spectroscopy of NS5A-6HIS and NS5A-Y93H-6HIS. Circular dichroism measurements of NS5A-6HIS or NS5A-Y93H-6HIS (10 μM) were carried out at 20°C. The average of 3 measurements is shown.

    Journal: PLoS ONE

    Article Title: Direct Binding of Ledipasvir to HCV NS5A: Mechanism of Resistance to an HCV Antiviral Agent

    doi: 10.1371/journal.pone.0122844

    Figure Lengend Snippet: Characterization of purified NS5A. (A) Coomassie staining of purified proteins. Recombinant His-tagged proteins were purified as described in Materials and Methods. 1 μg of NS5A-6HIS and NS5A-Y93H-6HIS were subjected to SDS-PAGE and the protein bands were visualized by Coomassie Blue stain. Molecular weights for protein standards are indicated. (B) Phosphorylation sites determined by mass spectrometry of purified NS5A-6HIS and NS5A-Y93H-6HIS. Phosphorylation sites identified in NS5A-6HIS are shaded green; sites identified in NS5A-Y93H-6HIS are shaded red; and sites identified in both proteins are shaded cyan. (C) Analytical ultracentrifugation analysis of NS5A-6HIS, NS5A-Y93H-6HIS, and NS5A-domain 1. For NS5A-6HIS and NS5A-Y93H-6HIS, analytical ultracentrifugation was performed in 25 mM Tris pH 7.5, 150 mM NaCl, 0.01% NaN 3 , 0.5 mM TCEP, 2 μg/ml Leupeptin, 0.02% C12E8, and 21.1% (v/v) D 2 O. Sedimentation velocity analysis was performed at 42,000 rpm for two protein concentrations (3.3 and 6.6 μM). For NS5A-domain 1, analytical ultracentrifugation was performed in 25 mM Tris pH 8.0, 250 mM NaCl, 10% glycerol, and 0.5% DMSO. Sedimentation velocity analysis was performed at 48,000 rpm for 4 protein concentrations (7.5, 12, 15, 30 μM). Representative traces are shown for each protein. (D) Circular dichroism spectroscopy of NS5A-6HIS and NS5A-Y93H-6HIS. Circular dichroism measurements of NS5A-6HIS or NS5A-Y93H-6HIS (10 μM) were carried out at 20°C. The average of 3 measurements is shown.

    Article Snippet: The PCR fragment was digested with Xba1 and EcoRV and ligated into pcDNA3.1(-) (Invitrogen). pcDNA3-NS5A-Y93H was generated by site-directed mutagenesis (Stratagene). pTK-NS5A was constructed by digesting pcDNA3-NS5A with Xba1 and EcoRV to isolate a 1.5 kb fragment encoding NS5A.

    Techniques: Purification, Staining, Recombinant, SDS Page, Mass Spectrometry, Sedimentation, Spectroscopy