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    New England Biolabs idez
    A , Schematic outlining <t>IdeZ</t> cleavage of IgG below the hinge region yielding multiple F(ab’) 2 and Fc fragments after reduction. B , Serum samples from mouse, dog, primate and human untreated (-) or treated (+) <t>with</t> <t>recombinant</t> IdeZ and analyzed by SDS-PAGE under reducing conditions. Gels were then stained with Coomassie blue. C , Pooled human IgG untreated (-) or treated (+) with recombinant GST-IdeZ or commercial standard IdeZ (NEB) and analyzed by SDS-PAGE under reducing conditions. Gels were then stained with Coomassie blue. IgG was cleaved by GST-IdeZ and IdeZ into multiple fragments as indicated. D , Mice were injected intraperitoneally first with pooled human IgG, following which they were injected intravenously 24 hours later with PBS (-) or recombinant GST-IdeZ (1 mg/kg) (+). Blood samples were taken 72 hours post injection and analyzed by SDS-PAGE under reducing conditions with immunoblotting. IgG was probed with Fab (top panel) and Fc (bottom panel) specific antibodies. E , Experimental timeline of in vivo GST-IdeZ dose optimization experiment. Mice were injected with pooled human IgG followed 24 hrs later with no injection or injection with 3 different doses of GST-IdeZ. Blood serum samples were collected 72 hours post GST-IdeZ. Sac., sacrifice followed by tissue harvest. Serum samples of PBS control ( F ), 0.25 mg/kg ( G ), 1 mg/kg ( H ), and 2.5 mg/kg ( I ) GST-IdeZ injected mice were analyzed by SDS-PAGE under reducing conditions and probed with human IgG specific antibodies to analyze IgG cleavage.
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    A , Schematic outlining IdeZ cleavage of IgG below the hinge region yielding multiple F(ab’) 2 and Fc fragments after reduction. B , Serum samples from mouse, dog, primate and human untreated (-) or treated (+) with recombinant IdeZ and analyzed by SDS-PAGE under reducing conditions. Gels were then stained with Coomassie blue. C , Pooled human IgG untreated (-) or treated (+) with recombinant GST-IdeZ or commercial standard IdeZ (NEB) and analyzed by SDS-PAGE under reducing conditions. Gels were then stained with Coomassie blue. IgG was cleaved by GST-IdeZ and IdeZ into multiple fragments as indicated. D , Mice were injected intraperitoneally first with pooled human IgG, following which they were injected intravenously 24 hours later with PBS (-) or recombinant GST-IdeZ (1 mg/kg) (+). Blood samples were taken 72 hours post injection and analyzed by SDS-PAGE under reducing conditions with immunoblotting. IgG was probed with Fab (top panel) and Fc (bottom panel) specific antibodies. E , Experimental timeline of in vivo GST-IdeZ dose optimization experiment. Mice were injected with pooled human IgG followed 24 hrs later with no injection or injection with 3 different doses of GST-IdeZ. Blood serum samples were collected 72 hours post GST-IdeZ. Sac., sacrifice followed by tissue harvest. Serum samples of PBS control ( F ), 0.25 mg/kg ( G ), 1 mg/kg ( H ), and 2.5 mg/kg ( I ) GST-IdeZ injected mice were analyzed by SDS-PAGE under reducing conditions and probed with human IgG specific antibodies to analyze IgG cleavage.

    Journal: bioRxiv

    Article Title: Rescuing AAV gene transfer from antibody neutralization with an IgG-degrading enzyme

    doi: 10.1101/2020.05.12.092122

    Figure Lengend Snippet: A , Schematic outlining IdeZ cleavage of IgG below the hinge region yielding multiple F(ab’) 2 and Fc fragments after reduction. B , Serum samples from mouse, dog, primate and human untreated (-) or treated (+) with recombinant IdeZ and analyzed by SDS-PAGE under reducing conditions. Gels were then stained with Coomassie blue. C , Pooled human IgG untreated (-) or treated (+) with recombinant GST-IdeZ or commercial standard IdeZ (NEB) and analyzed by SDS-PAGE under reducing conditions. Gels were then stained with Coomassie blue. IgG was cleaved by GST-IdeZ and IdeZ into multiple fragments as indicated. D , Mice were injected intraperitoneally first with pooled human IgG, following which they were injected intravenously 24 hours later with PBS (-) or recombinant GST-IdeZ (1 mg/kg) (+). Blood samples were taken 72 hours post injection and analyzed by SDS-PAGE under reducing conditions with immunoblotting. IgG was probed with Fab (top panel) and Fc (bottom panel) specific antibodies. E , Experimental timeline of in vivo GST-IdeZ dose optimization experiment. Mice were injected with pooled human IgG followed 24 hrs later with no injection or injection with 3 different doses of GST-IdeZ. Blood serum samples were collected 72 hours post GST-IdeZ. Sac., sacrifice followed by tissue harvest. Serum samples of PBS control ( F ), 0.25 mg/kg ( G ), 1 mg/kg ( H ), and 2.5 mg/kg ( I ) GST-IdeZ injected mice were analyzed by SDS-PAGE under reducing conditions and probed with human IgG specific antibodies to analyze IgG cleavage.

    Article Snippet: All in vitro activity assays with recombinant GST-IdeZ or IdeZ (NEB Catalog #P0770S) (1ug per reaction) were performed for 3 h at 37°C and serum samples were diluted 50x in PBS prior to analysis by SDS-PAGE.

    Techniques: Recombinant, SDS Page, Staining, Injection, Western Blot, In Vivo

    A , Experimental timeline of IgG, IdeZ and AAV8 or AAV9-Luc injections. Sac., sacrifice followed by tissue harvest. Mice were injected intraperitoneally with pooled human IgG. The same mice were injected intravenously 24 hours later with PBS or recombinant GST-IdeZ (2.5 mg/kg). AAV8-Luc or AAV9-Luc was injected 72 hours post IdeZ at a dose of 1 × 10 13 vg/kg. Luciferase transgene expression levels were analyzed 4 weeks post-injection in the liver; AAV8 ( B,C ); AAV9 ( D,E ). Luciferase expression levels were normalized for total tissue protein concentration and represented as log relative luminescence units per gram of tissue (log RLU/g tissue). Each dot represents the average of a technical duplicate from a single animal. Biodistribution of AAV8 and AAV9 Luc vector genomes in the liver; AAV8 ( F,G ); AAV9 ( H,I ). Vector genome copy numbers per cell were calculated by normalizing Luc copy numbers to copies of the Lamin B2 housekeeping gene and represented as log vg/cell. Each dot represents a technical duplicate from a single animal, and the dash represents the mean value. (F=female, M=male). J , Schematic demonstrating experimental timeline of IdeZ and AAV9-Luc injections in NHPs. AAV9 seropositive NHP M16558 (n=1) was administered IdeZ (0.5 mg/kg) via intravenous bolus injection on Day 0. AAV9-Luc was administered via intravenous bolus injection 72 hrs post-IdeZ injection at a dose of 5 × 10 12 vg/kg. K , NHP serum samples were analyzed by SDS-PAGE under reducing conditions and probed with Fc specific antibodies. L , Luciferase transgene expression levels were analyzed 4 weeks post-injection in the liver of NHPs. Luciferase expression levels were normalized for total tissue protein concentration and represented as log relative luminescence units per gram of tissue (Log RLU/g tissue). Each dot represents a single experiment of an individual liver lobe from a single animal. M , Biodistribution of AAV9 Luc vector genomes in the liver of NHPs. Vector genome copy numbers per ng of total extracted DNA were calculated and represented as log vg/ng DNA. Each dot represents a technical duplicate experiment of individual liver slices from a single animal and the dash represents the mean value. Significance was determined by one-way ANOVA with Tukey’s post-test. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001

    Journal: bioRxiv

    Article Title: Rescuing AAV gene transfer from antibody neutralization with an IgG-degrading enzyme

    doi: 10.1101/2020.05.12.092122

    Figure Lengend Snippet: A , Experimental timeline of IgG, IdeZ and AAV8 or AAV9-Luc injections. Sac., sacrifice followed by tissue harvest. Mice were injected intraperitoneally with pooled human IgG. The same mice were injected intravenously 24 hours later with PBS or recombinant GST-IdeZ (2.5 mg/kg). AAV8-Luc or AAV9-Luc was injected 72 hours post IdeZ at a dose of 1 × 10 13 vg/kg. Luciferase transgene expression levels were analyzed 4 weeks post-injection in the liver; AAV8 ( B,C ); AAV9 ( D,E ). Luciferase expression levels were normalized for total tissue protein concentration and represented as log relative luminescence units per gram of tissue (log RLU/g tissue). Each dot represents the average of a technical duplicate from a single animal. Biodistribution of AAV8 and AAV9 Luc vector genomes in the liver; AAV8 ( F,G ); AAV9 ( H,I ). Vector genome copy numbers per cell were calculated by normalizing Luc copy numbers to copies of the Lamin B2 housekeeping gene and represented as log vg/cell. Each dot represents a technical duplicate from a single animal, and the dash represents the mean value. (F=female, M=male). J , Schematic demonstrating experimental timeline of IdeZ and AAV9-Luc injections in NHPs. AAV9 seropositive NHP M16558 (n=1) was administered IdeZ (0.5 mg/kg) via intravenous bolus injection on Day 0. AAV9-Luc was administered via intravenous bolus injection 72 hrs post-IdeZ injection at a dose of 5 × 10 12 vg/kg. K , NHP serum samples were analyzed by SDS-PAGE under reducing conditions and probed with Fc specific antibodies. L , Luciferase transgene expression levels were analyzed 4 weeks post-injection in the liver of NHPs. Luciferase expression levels were normalized for total tissue protein concentration and represented as log relative luminescence units per gram of tissue (Log RLU/g tissue). Each dot represents a single experiment of an individual liver lobe from a single animal. M , Biodistribution of AAV9 Luc vector genomes in the liver of NHPs. Vector genome copy numbers per ng of total extracted DNA were calculated and represented as log vg/ng DNA. Each dot represents a technical duplicate experiment of individual liver slices from a single animal and the dash represents the mean value. Significance was determined by one-way ANOVA with Tukey’s post-test. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001

    Article Snippet: All in vitro activity assays with recombinant GST-IdeZ or IdeZ (NEB Catalog #P0770S) (1ug per reaction) were performed for 3 h at 37°C and serum samples were diluted 50x in PBS prior to analysis by SDS-PAGE.

    Techniques: Injection, Recombinant, Luciferase, Expressing, Protein Concentration, Plasmid Preparation, SDS Page

    A , Schematic demonstrating experimental timeline of human serum, IdeZ and AAV9-Luc injections. 18 human serum samples were tested for their ability to neutralize AAV9 transduction in the liver. Two mice per human serum sample were utilized for the study and both mice were injected intraperitoneally with human serum. Mice were then injected intravenously 72 hours later with PBS (black bars) or recombinant GST-IdeZ (2.5 mg/kg, grey bars) and subsequently injected intravenously 72 hrs post-IdeZ treatment with AAV9-Luc (1 × 10 13 vg/kg). Liver transduction levels were analyzed 4 weeks post-injection. Sac., sacrifice followed by tissue harvest. B , Luciferase transgene expression levels were analyzed 4 weeks post-injection in the liver of passively immunized mice treated with PBS (black) or prophylactically with IdeZ (grey). Transduction levels were normalized to non-immunized mice that were injected with AAV9-Luc at the same dose and represented as percentage of control. Each bar represents the average of a technical duplicate from a single animal. C , Relative liver transduction efficiency of AAV9-Luc in the entire cohort of mice immunized with human sera treated with PBS control (white) or IdeZ (grey). Biodistribution of AAV9 vector genomes in the liver for mice passively immunized with individual human serum samples ( D ) and the entire cohort ( E ). Vector genome copy numbers per cell were calculated based on normalization to copies of the Lamin B2 housekeeping gene. Each bar represents the average of a technical duplicate from a single animal. Significance was determined by the nonparametric Mann-Whitney rank test. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.

    Journal: bioRxiv

    Article Title: Rescuing AAV gene transfer from antibody neutralization with an IgG-degrading enzyme

    doi: 10.1101/2020.05.12.092122

    Figure Lengend Snippet: A , Schematic demonstrating experimental timeline of human serum, IdeZ and AAV9-Luc injections. 18 human serum samples were tested for their ability to neutralize AAV9 transduction in the liver. Two mice per human serum sample were utilized for the study and both mice were injected intraperitoneally with human serum. Mice were then injected intravenously 72 hours later with PBS (black bars) or recombinant GST-IdeZ (2.5 mg/kg, grey bars) and subsequently injected intravenously 72 hrs post-IdeZ treatment with AAV9-Luc (1 × 10 13 vg/kg). Liver transduction levels were analyzed 4 weeks post-injection. Sac., sacrifice followed by tissue harvest. B , Luciferase transgene expression levels were analyzed 4 weeks post-injection in the liver of passively immunized mice treated with PBS (black) or prophylactically with IdeZ (grey). Transduction levels were normalized to non-immunized mice that were injected with AAV9-Luc at the same dose and represented as percentage of control. Each bar represents the average of a technical duplicate from a single animal. C , Relative liver transduction efficiency of AAV9-Luc in the entire cohort of mice immunized with human sera treated with PBS control (white) or IdeZ (grey). Biodistribution of AAV9 vector genomes in the liver for mice passively immunized with individual human serum samples ( D ) and the entire cohort ( E ). Vector genome copy numbers per cell were calculated based on normalization to copies of the Lamin B2 housekeeping gene. Each bar represents the average of a technical duplicate from a single animal. Significance was determined by the nonparametric Mann-Whitney rank test. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.

    Article Snippet: All in vitro activity assays with recombinant GST-IdeZ or IdeZ (NEB Catalog #P0770S) (1ug per reaction) were performed for 3 h at 37°C and serum samples were diluted 50x in PBS prior to analysis by SDS-PAGE.

    Techniques: Transduction, Injection, Recombinant, Luciferase, Expressing, Plasmid Preparation, MANN-WHITNEY