- 711375 - Invitrogen Antibodies PARP1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: CRISPR-Cas9 mediated genome editing ofPARP1 (as confirmed by next generation sequencing) was achieved by using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR978664_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Fig (a) Western blot analysis of PARP1 was performed by loading 30 µg of HeLa Wild Type (Lane 1), HeLa Wild Type treated with Staurosporin (1 µM for 16 hrs) (Lane 2), HeLa Cas9 (Lane 3), HeLa Cas9 treated with Staurosporin (1 µM for 16hrs) (Lane 4), HeLa Cas9 cells transduced with PARP1 Lentiviral sgRNA (Lane 5) and HeLa Cas9 cells transduced with PARP1 Lentiviral sgRNA treated with Staurosporin (1 µM for 16 hrs) (Lane 6) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-PARP1 Recombinant Polyclonal Antibody (8HCLC) (Product # 711375) using 1:500 dilution and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036 1:2,500 dilution).Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal in sgRNA transduced cells using the LentiArray™ CRISPR product line confirms that antibody is specific to PARP1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on Nuclear cell extracts (30 µg lysate) of Jurkat (Lane 1), Jurkat treated with Staurosporine (1uM overnight) (Lane 2), NTERA-2 (Lane 3), NTERA-2 treated with Staurosporine (1uM overnight0 (Lane 4), SH-SY5Y (Lane 5) and SH-SY5Y treated with Staurosporine (1uM overnight) (Lane 6). The blots were probed with Anti-Cleaved PARP Recombinant Rabbit Polyclonal Antibody (Product # 711375, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 89 kDa band corresponding to Cleaved PARP was observed. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: CRISPR-Cas9 mediated genome editing ofPARP1 (as confirmed by next generation sequencing) was achieved by using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR978664_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Fig (a) Western blot analysis of PARP1 was performed by loading 30 µg of HeLa Wild Type (Lane 1), HeLa Wild Type treated with Staurosporin (1 µM for 16 hrs) (Lane 2), HeLa Cas9 (Lane 3), HeLa Cas9 treated with Staurosporin (1 µM for 16hrs) (Lane 4), HeLa Cas9 cells transduced with PARP1 Lentiviral sgRNA (Lane 5) and HeLa Cas9 cells transduced with PARP1 Lentiviral sgRNA treated with Staurosporin (1 µM for 16 hrs) (Lane 6) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-PARP1 Recombinant Polyclonal Antibody (8HCLC) (Product # 711375) using 1:500 dilution and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036 1:2,500 dilution).Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal in sgRNA transduced cells using the LentiArray™ CRISPR product line confirms that antibody is specific to PARP1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: For immunofluorescence analysis, Staurosporine (1 uM, 3 h) treated HeLa cells were fixed and permeabilized for detection of endogenous Cleaved PARP using Anti-Cleaved PARP Recombinant Rabbit Polyclonal Antibody (Product # 711375, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of Cleaved PARP protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating nuclear localization of Cleaved PARP. Panel e) shows no signal in untreated cells, demonstrating antibody specificity. Panel f)represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

711375

Antibody data