Brd4 antibody

Manufactured by Fortis Life Sciences

The BRD4 antibody is a laboratory research tool used to detect and study the expression of the BRD4 protein, which is a member of the bromodomain and extra-terminal (BET) protein family. BRD4 plays a role in regulating gene transcription and is involved in various cellular processes. The antibody can be used in techniques such as Western blotting, immunohistochemistry, and immunofluorescence to analyze BRD4 expression and localization in biological samples.

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Lab products found in correlation

Nbp2 16550, by Santa Cruz Biotechnology (2 mentions) Rxrα antibody, by Proteintech (2 mentions) 6e10 antibody, by BioLegend (1 mentions) Lc3a b antibody, by Cell Signaling Technology (1 mentions) β actin antibody, by Merck Group (1 mentions) Arv825, by MedChemExpress (1 mentions) Ecl solution, by Thermo Fisher Scientific (1 mentions) Vinculin, by Santa Cruz Biotechnology (1 mentions) 4 hydroxytamoxifen 4 oht, by Merck Group (1 mentions) Lamin b antibody, by Santa Cruz Biotechnology (1 mentions) Tamoxifen, by Merck Group (1 mentions) Control rabbit igg, by R&D Systems (1 mentions) Magnify chip system, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Anti-BRD4 antibody (6 citations) Anti-BRD4 (A301-985A-M) antibody (2 citations)

8 protocols using brd4 antibody

Targeted Detection of APP and Tau Markers

Cited 2 times

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Chemicals, including ARV-825 and JQ1, were from Medchemexpress (Catalog #s: HY-16954 and HY-13030, respectively) and their stocks were in DMSO. The G12A antibody was used to detect APP full length and C-terminal fragments (APP-CTFs) (rabbit polyclonal, clone C7 targeting amino acid residues 732–751 of APP751, custom made by Thermo Fisher Scientific) and were previously reported (57 (link), 58 (link), 59 (link)). The sAPPβ antibody was purchased from IBL (Catalog #: 18975). The 6E10 antibody (BioLegend) is a monoclonal antibody (mAb) reactive to amino acid residues 1–16 of Aβ from the N-terminal sequence and can detect sAPPα in medium. The nicastrin (NCT) antibody (Catalog #: 5665), BACE1 antibody (Catalog #: 5606), and LC3 a/b antibody (Catalog #: 12741) were purchased from Cell Signaling. The BRD4 antibody was from BETHYL (Catalog #: A700-004-T). The β-actin antibody was from Sigma and used as a protein control. The antibody for pTau (S396) was from Cell Signaling (Catalog #: 9632). The HRP-conjugated secondary antibodies (anti-mouse and anti-rabbit) were from Pierce. The dilution factors for primary and secondary antibodies were 1: 1000 and 1: 10,000, respectively.

Zhang S., Bai P., Lei D., Liang Y., Zhen S., Bakiasi G., Pang H., Choi S.H., Wang C., Tanzi R.E, & Zhang C. (2022). Degradation and inhibition of epigenetic regulatory protein BRD4 exacerbate Alzheimer’s disease-related neuropathology in cell models. The Journal of Biological Chemistry, 298(4), 101794.

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Western Blot Analysis of Cell Lysates

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Cultured cells were washed with 1 ml phosphate-buffered saline (PBS) and 1 million cells were counted. Cells were resuspended in 150 μl RIPA buffer [150 mM NaCl, 50 mM Tris–Cl (pH 8.0), 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS) and protease inhibitor cocktail] and then incubated for 10 min at ice. These whole-cell lysates were centrifuged for 10 min at 4°C. The supernatant was mixed with 50 μl of 5× sample buffer [Bromophenol Blue 0.25%, DTT 0.5 M, glycerol 50%, SDS 10%, 0.25 M Tris–Cl (pH 6.8)]. The lysed sample was boiled at 95°C for 10 min. Each sample was loaded and separated by 9–10% SDS-PAGE gel electrophoresis and transferred onto nitrocellulose membranes at 4°C for 3 h. The membrane was blocked in 5% skimmed milk in Tris-buffered saline and Tween 20 (TBST) for 30 min. The blocked membrane was overnight incubated with the corresponding primary antibodies at 4°C. The membrane was washed with TBST and incubated with secondary antibodies for 40 min at room temperature. The membrane was washed with TBST and visualized using ECL solution (REF# 186309716, Thermo Scientific). The primary antibodies were VINCULIN (sc-73614, Santa Cruz), TUBULIN (sc-23948, Santa Cruz), EVI1 (2593S, Cell Signaling), WNT7A (10605-1-AP, Protein Tech) and BRD4 antibody (A301-985A50, Bethyl Laboratories).

Kim H.R., Yim J., Yoo H.B., Lee S.E., Oh S., Jung S., Hwang C.I., Shin D.M., Kim T., Yoo K.H., Kim Y.S., Lee H.W, & Roe J.S. (2021). EVI1 activates tumor-promoting transcriptional enhancers in pancreatic cancer. NAR Cancer, 3(2), zcab023.

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BRD4 Chromatin Immunoprecipitation Assay

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Kasumi-1 cells were seeded at 0.5 × 10⁶/ml at the day of experiment, treated with DMSO, 250 nM JQ1, and 125 nM MS417 for 1 hr. DNA/protein was crosslinked with 1% formaldehyde at room temperature for 10 min and crosslinking was terminated by 1.25 mM glycine. Crosslinked cells were washed twice with cold PBS, lysed with 1% SDS lysis buffer, and sonicated. 100 μl of sonicated chromatin was transferred to 900 μl cold dilution buffer, BRD4 antibody (Bethyl Laboratories, Montgomery, TX) and protein G magnetic beads were added, and reactions were incubated at 4° C overnight. Immunoprecipitated protein/DNA complexes were washed and crosslinking was reversed before DNA isolation. 4 μl of DNA was used in each quantitative PCR to assess BRD4 enrichment. Data were calculated relative to inputs and a transcriptionally inactive region for normalization and background reduction.

Zhao Y., Liu Q., Acharya P., Stengel K.R., Sheng Q., Zhou X., Kwak H., Fischer M.A., Bradner J.E., Strickland S.A., Mohan S.R., Savona M.R., Venters B.J., Zhou M.M., Lis J.T, & Hiebert S.W. (2016). High Resolution Mapping of RNA Polymerases Identifies Mechanisms of Sensitivity and Resistance to BET Inhibitors in t(8;21) AML. Cell reports, 16(7), 2003-2016.

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Liver ChIP Assay for FXR, RXRα, and BRD4

Cited 1 time

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Liver ChIP assay was performed as described previously (Jung et al., 2020 (link); Seok et al., 2018 (link)). Briefly, chromatin extracts were prepared from FXR floxed and FXR-LKO mouse livers after treatment of the mice with GW4064 which was followed by preclearing and immunoprecipitation using control IgG or FXR antibody (Novus Biologicals, NBP2–16550; Santa Cruz, sc-25309), RXRα antibody (Proteintech, catalog no. 21218-1-AP), or BRD4 antibody (Bethyl Laboratories, catalog #A301–985A50). Enrichment in chromatin precipitates of gene sequences was measured by qRCR using primers listed (Supplementary Table S6).

Chen J., Wang R., Xiong F., Sun H., Kemper B., Li W, & Kemper J.K. (2024). Hammerhead-type FXR agonists induce an eRNA FincoR that ameliorates nonalcoholic steatohepatitis in mice. bioRxiv.

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BRD4 and Cyclin T1 Affinity Capture

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HEK293 cell lysate was incubated with either biotinylated UMB-136 or biotin in DMSO at 4°C overnight with orbital rocking. MyOneTM Streptavidin T1 DynabeadsTM (Invitrogen) were added to the mixture which was then incubation for 2 h. Beads were washed and then boiled to elute precipitated proteins, which were subjected to immunoblotting using BRD4 antibody (Bethyl Laboratories, Inc.) or BRD2 antibody (Santa Cruz).
Tat-Flag expressing HeLa cells were treated with UMB-136 (2.5 μM) or DMSO for 24 h, then incubated with Magnetic Flag Conjugated beads (Clontch) at 4°C for overnight with orbital rocking. Beads were washed and boiled, then immunoblotting was performed using Cyclin T1 antibody (Santa Cruz).

Huang H., Liu S., Jean M., Simpson S., Huang H., Merkley M., Hayashi T., Kong W., Rodríguez-Sánchez I., Zhang X., Yosief H.O., Miao H., Que J., Kobie J.J., Bradner J., Santoso N.G., Zhang W, & Zhu J. (2017). A Novel Bromodomain Inhibitor Reverses HIV-1 Latency through Specific Binding with BRD4 to Promote Tat and P-TEFb Association. Frontiers in Microbiology, 8, 1035.

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Liver ChIP Assay for FXR and RXRα Binding

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Liver ChIP assay was performed as described previously (Jung et al., 2020 (link); Seok et al., 2018 (link)). Briefly, chromatin extracts were prepared from Fxr-Flox and Fxr-LKO mouse livers after treatment of the mice with GW4064 which was followed by preclearing and immunoprecipitation using control IgG or FXR antibody (Novus Biologicals, NBP2-16550; Santa Cruz, sc-25309), RXRα antibody (Proteintech, catalog no. 21218-1-AP), or BRD4 antibody (Bethyl Laboratories, catalog #A301-985A50). Enrichment in chromatin precipitates of gene sequences was measured by qPCR using primers listed (Supplementary file 5).

Chen J., Wang R., Xiong F., Sun H., Kemper B., Li W, & Kemper J. (2024). Hammerhead-type FXR agonists induce an enhancer RNA Fincor that ameliorates nonalcoholic steatohepatitis in mice. eLife, 13, RP91438.

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Antibody Validation and Chemical Compound Usage

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All antibodies were mouse specific unless otherwise noted. Rabbit anti-mouse PU.1 and MITF antibodies used for ChIP and WB have been described previously.^{20 (link)} BRD4 antibody was purchased from Bethyl Laboratories (Montgomery, TX). LAMIN B antibody was purchased from Santa Cruz Biotechnology (Dallas, TX). Anti-rabbit and anti-goat secondary antibodies (LI-COR Biosciences, Lincoln, NE) were used for WB. Tamoxifen and 4-hydroxyTamoxifen (4-OHT) were purchased from Sigma-Aldrich (St. Louis, MO). Tamoxifen was dissolved in corn oil with 5% ethanol and 4-OHT was dissolved in 95% ethanol. The bromodomain inhibitor JQ1 was a kind gift from Dr. Jay Bradner (Harvard Medical School).^{37 (link)}

Carey H.A., Hildreth BE I.I.I., Geisler J.A., Nickel M.C., Cabrera J., Ghosh S., Jiang Y., Yan J., Lee J., Makam S., Young N.A., Valiente G.R., Jarjour W.N., Huang K., Rosol T.J., Toribio R.E., Charles J.F., Ostrowski M.C, & Sharma S.M. (2018). Enhancer variants reveal a conserved transcription factor network governed by PU.1 during osteoclast differentiation. Bone Research, 6, 8.

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ChIP-qPCR Profiling of EWSR1 Loci

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A ChIP assay was performed by the Magnify ChIP system (Invitrogen-Life Technologies Inc.) using 5 mg of BRD4 antibody (BethylLab) or control rabbit IgG (R & D Systems). Real-time PCR (Bio-Rad) was performed on fragmented DNA using specific primers for the EWSR1 loci. Primers were designed to amplify sites within each gene locus based on the H3K27 acetylation level. The levels of enrichment of the H3K27Ac histone mark across the genome has been determined by a ChIP-seq assay on seven cell lines from ENCODE web resources. We used the following primers: EWSR1–1 sense 5′-CCGTAAACCTCCTCCTGCAT-3′ and anti-sense 5′-AAGCCCTTCACCCTTGCTAA-3′; EWSR1–2 sense 5′-GCAGTTGTTCTAGTCCGGGT-3′ and anti-sense 5′-CCGCAACTCTTGTCCCAGTC-3′; EWSR1–3 sense 5′-AAGACTGAGTGGAGTTGCCG-3′ and anti-sense 5′-GAAGATTCCAGAACCGGCCC-3′. For the ChIP-qPCR, error bars show standard deviation for n = 3 measurements from representative experiments. Each assay was performed in triplicate.

Jacques C., Lamoureux F., Baud’huin M., Calleja L.R., Quillard T., Amiaud J., Tirode F., Rédini F., Bradner J.E., Heymann D, & Ory B. (2016). Targeting the epigenetic readers in Ewing Sarcoma inhibits the oncogenic transcription factor EWS/Fli1. Oncotarget, 7(17), 24125-24140.

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