- PA5-30263 - Invitrogen Antibodies BRD3 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of BRD3 using 20 µg of HeLa nucleus lysate. Samples were loaded onto a 7.5% SDS-PAGE gel and probed with a BRD3 polyclonal antibody (Product # PA5-30263) at a dilution of 1:1000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot analysis of BRD3 was performed by separating 30 µg of HeLa whole cell and nuclear extracts by 5% SDS-PAGE. Proteins were transferred to a membrane and probed with a BRD3 Polyclonal Antibody (Product # PA5-30263) at a dilution of 1:1000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot using BRD3 Polyclonal Antibody (Product # PA5-30263). Sample (20 µg of whole cell lysate). Lane A: mouse Liver. 7.5% SDS PAGE. BRD3 Polyclonal Antibody (Product # PA5-30263) diluted at 1:20,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot using BRD3 Polyclonal Antibody (Product # PA5-30263). HeLa whole cell and nuclear extracts (30 µg) were separated by 5% SDS-PAGE, and the membrane was blotted with BRD3 Polyclonal Antibody (Product # PA5-30263) diluted at 1:1,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-BRD3 Polyclonal Antibody (Product # PA5-30263) and a 60 kDa band corresponding to BRD3 was observed in the cell lines tested. Whole cell extracts (30 µg lysate) of NTERA-2 cl.D1 (Lane 1), A549 (Lane 2), SH-SY5Y (Lane 3), HeLa (Lane 4) (also an uncharacterized band (*) was observed in Lane 4 at ~50 kDa) and IMR32 (Lane 5) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX). 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 conjugate (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: BRD3 Polyclonal Antibody detects BRD3 protein at nucleus by immunofluorescent analysis. Sample: HeLa cells were fixed in 4% paraformaldehyde at RT for 15 min. Green: BRD3 stained by BRD3 Polyclonal Antibody (Product # PA5-30263) diluted at 1:500. Red: phalloidin, a cytoskeleton marker, diluted at 1:200. Scale bar= 10 µm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of BRD3 was performed using 70% confluent log phase NTERA-2 cl.D1 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 BRD3 Polyclonal Antibody (Product # PA5-30263) 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 conjugate (Product # A27034) 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 predominantly nuclear localization. Panel e represents 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: BRD3 Polyclonal Antibody detects BRD3 protein at nucleus by immunohistochemical analysis. Sample: Paraffin-embedded mouse testis. BRD3 stained by BRD3 Polyclonal Antibody (Product # PA5-30263) diluted at 1:500. Antigen Retrieval: Citrate buffer, pH 6.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Chromatin Immunoprecipitation (ChIP) assay of endogenous BRD3 protein using Anti-BRD3 Antibody: ChIP was performed using Anti-BRD3 Rabbit polyclonal Antibody (Product # PA5-30263, 5 µg) on sheared chromatin from NTERA 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 CCND1 and MYC 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.

PA5-30263