- 702872 - Invitrogen Antibodies BRD8 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-BRD8 Recombinant Rabbit Monoclonal Antibody (Product # 702872) and a ~135 kDa band corresponding to BRD8 was observed across cell lines tested. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), HCT 116 (Lane 2) were electrophoresed using Novex® NuPAGE® 10% Bis-Tris gel (Product # NP0302BOX). Resolved proteins were then transferred onto a nitrocellulose membrane by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:500 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 1:4000 dilution). 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: Knockdown of BRD8 was achieved by transfecting HCT 116 cells with BRD8 specific siRNA (Silencer® select Product # S21425 and S21424). Western blot analysis (Fig a) was performed using whole cell extracts from BRD8 knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with Anti-BRD8 Recombinant Rabbit Monoclonal Antibody (Product # 702872, 1:500 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in the histogram (Fig b). Loss of signal upon siRNA mediated knockdown confirms that antibody is specific to BRD8.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of BRD8 was performed by loading 100 ng of whole cell lysates from Control (Lane 1 and 2) and BRD8 Knock down cells (lane 3 and 4) in RIPA Lysis and Extraction buffer (Product # 89900) and Page Ruler Plus Protein Ladder (Product # 26619) onto a 30% Acrylamide/Bis gel. Proteins were transferred to membrane and blocked in 5% milk for one hour at room temperature. BRD8 was detected at approximately 140 kDa using a BRD8 recombinant monoclonal antibody (Product # 702872) at a dilution of 1:500 in 5% milk overnight at 4°C on a rocking platform, followed by a goat anti-rabit IgG-HRP secondary antibody at a dilution of 1:5,000 for one hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34076) and the myECL Imager (Product # 62236). Data courtesy of Wei Xu’s laboratory at the University of Wisconsin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: For immunofluorescence analysis, HCT 116 cells were fixed and permeabilized for detection of endogenous BRD8 using Anti-BRD8 Recombinant Rabbit Monoclonal Antibody (Product # 702872, 1:100 dilution) and labeled with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 conjugate (Product # A32731, 1:2000). Panel a) shows representative cells that were stained for detection and localization of BRD8 protein (green), Panel b) is stained for nuclei (blue) using ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). Panel c) represents cytoskeletal F-actin staining using Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating nuclear localization of BRD8. 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 BRD8 protein using Anti-BRD8 Antibody: ChIP was performed using Anti-BRD8 Recombinant Rabbit Monoclonal Antibody (Product # 702872, 5 µg) on sheared chromatin from 2 million HCT116 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 MMP13 and MMP2 transcriptional start site, GAPDH 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.

702872