- PA5-29158 - Invitrogen Antibodies ERCC1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of ERCC1 using 30 µg of HeLa lysate. Samples were loaded onto a 12% SDS-PAGE gel and probed with an ERCC1 polyclonal antibody (Product # PA5-29158) at a dilution of 1:1000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of ERCC1 was achieved by transfecting U-87 MG cells with ERCC1 specific siRNAs (Silencer® select Product # s4784, s478). Western blot analysis (Fig. a) was performed using modified whole cell extracts (1% SDS) from the ERCC1 knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blots were probed with Anti-ERCC1 Polyclonal Antibody (Product # PA5-29158, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this Western Blot is shown in histogram (Fig. b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to ERCC1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-ERCC1 Polyclonal Antibody (Product # PA5-29158) and 36 kDa band corresponding to ERCC1 was observed across the cell lines tested. Modified whole cell extracts (1% SDS) (30 µg lysate) of U-87 MG (Lane1), Hep G2 (Lane 2), HeLa (Lane 3) and A-431 (Lane 4) were electrophoresed using Novex® NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0302BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: ERCC1 Polyclonal Antibody detects ERCC1 protein at nucleus by immunofluorescent analysis. Sample: A549 cells were fixed in 4% paraformaldehyde at RT for 15 min. Green: ERCC1 protein stained by ERCC1 Polyclonal Antibody (Product # PA5-29158) diluted at 1:1,000. Blue: Hoechst 33342 staining. Scale bar = 10 µm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of ERCC1 was performed using 70% confluent log phase U-87 MG cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with ERCC1 Polyclonal Antibody (Product # PA5-29158) at 1:100 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

PA5-29158