- PA5-17359 - Invitrogen Antibodies TFAP2A antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of AP-2-alpha in extracts from A204 and DLD-1 cell lines using AP-2-alpha polyclonal antibody (Product # PA5-17359).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of AP2 alpha was achieved by transfecting SK-BR-3 cells with AP2 alpha specific siRNAs (Silencer® select Product # s14004, s14003). Western blot analysis (Fig. a) was performed using whole cell extracts from the AP2 alpha knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blots were probed with Anti-AP2 alpha Polyclonal Antibody (Product # PA5-17359, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP conjugate (Product # A27036, 0.25 ug/ml, 1:4000 dilution). Densitometric analysis of this Western Blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to AP alpha.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-AP2 alpha Polyclonal Antibody (Product # PA5-17359) and a 50 kDa band corresponding to AP2 alpha was observed across all the cell lines tested. Modified whole cell extracts (1% SDS) (30 µg lysate) of HeLa (Lane 1), SK-BR-3 (Lane 2), A-431 (Lane 3), PC-3 (Lane 4), LNCaP (Lane 5), MCF7 (Lane 6), MDA-MB-231 (Lane 7) and RAW 264.7 (Lane 8) were electrophoresed using Novex® NuPAGE® 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 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: Immunofluorescence analysis AP2 alpha was performed using 70% confluent log phase SK-BR-3 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 AP2 alpha Polyclonal Antibody (Product PA5-17359) at 1:100 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, Alexa Fluor 488 (Product # A28175) 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Chromatin Immunoprecipitation (ChIP) assay of endogenous AP2 alpha protein using Anti-AP2 alpha Antibody: ChIP was performed using Anti-AP2 alpha Polyclonal Antibody (Product # PA5-17359) 5 µg, on sheared chromatin from MCF-7 cells using the MAGnify ChIP System kit (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by qPCR using primers binding to transcriptional start site of ANKRD1, CDX2 promoter and SAT2 satellite repeats. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

PA5-17359