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82970
BRD4 Antibody Sampler Kit
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BRD4 Antibody Sampler Kit #82970

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Enhanced cross-linking and immunoprecipitation (eCLIP) was performed with RNA from K-562 cells and BRD4 (E2A7X) Rabbit mAb using a protocol based on the RBP-eCLIP method from Eclipsebio. The figure shows binding across the TBCD transcript. Data is kindly provided by the laboratory of Dr. Gene Yeo and used with permission.
Immunoprecipitation of BRD4 isoform C protein from 293T cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is BRD4 Isoform C (E1Q9N) Rabbit mAb. Western blot analysis was performed using BRD4 Isoform C (E1Q9N) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.
Confocal immunofluorescent analysis of HCT 116 cells, either mock transfected (left, high-expressing) or transfected with siRNA directed against BRD4 (right, low-expressing), using BRD4 Isoform C (E1Q9N) Rabbit mAb (green), DyLight 650 Phalloidin #12956 (red), and DAPI #4083 (blue).
Chromatin immunoprecipitations were performed with cross-linked chromatin from MV-4-11 cells and either BRD4 Isoform C (E1Q9N) Rabbit mAb or Normal Rabbit IgG #2729, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR, using human BCL2 promoter primers, SimpleChIP® Human c-Myc Intron 1 Primers #14905, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
Western blot analysis of extracts from RL, 293T, and Jurkat cells using BRD4 (E2A7X) Rabbit mAb.
Western blot analysis of extracts from various cell lines using BRD4 Isoform C (E1Q9N) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
Western blot analysis of extracts from 293T cells, either mock transfected (-) or transfected (+) with a construct expressing Myc/DDK-tagged full-length human BRD4 IsoB protein (hBRD4IsoB-Myc/DDK) or Myc/DDK-tagged full-length human BRD4 IsoC protein (hBRD4IsoC-Myc/DDK), using BRD4 (pan) (E1Y1P) Rabbit mAb (upper) and DYKDDDDK Tag (D6WB) Rabbit mAb (Binds to same epitope as Sigma's Anti-FLAG® M2 Antibody) #14793 (lower).
Chromatin immunoprecipitations were performed with cross-linked chromatin from untreated MV-4-11 cells and BRD4 (pan) (E1Y1P) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Library was prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figure shows binding across CCDC26 gene.
Immunoprecipitation of BRD4 from RL cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or BRD4 (E2A7X) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using BRD4 (E2A7X) Rabbit mAb.
Western blot analysis of extracts from 293T cells, transfected with control siRNA (-) or human BRD4 isoform C siRNA (hBRD4 Isoform C siRNA; +), using BRD4 Isoform C (E1Q9N) Rabbit mAb or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from 293T cells, mock transfected or transfected with BRD4 siRNA, and extracts from Jurkat, Caki-1, and NIH/3T3 cells using BRD4 (pan) (E1Y1P) Rabbit mAb (upper) or α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower). As expected, reduced signal is observed in 293T cells transfected with BRD4 siRNA.
Chromatin immunoprecipitations were performed with cross-linked chromatin from untreated MV-4-11 cells and BRD4 (pan) (E1Y1P) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Library was prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 8 (upper), including CCDC26 gene (lower).
Immunohistochemical analysis of paraffin-embedded human squamous cell carcinoma of the tonsil (left), normal kidney (middle) or normal prostate (right) using BRD4 (E2A7X) Rabbit mAb (top) or BRD4 Rabbit mAb (bottom). These two antibodies detect unique, non-overlapping epitopes on human BRD4. The similar staining patterns obtained with both antibodies help to confirm the specificity of the staining. 
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected (+) with constructs expressing Myc/DDK-tagged full-length human BRD4 isoform B (BRD4 Isoform B-Myc/DDK) or Myc/DDK-tagged full-length human BRD4 isoform C (BRD4 Isoform C-Myc/DDK), using BRD4 Isoform C (E1Q9N) Rabbit mAb (upper) or DYKDDDDK Tag (D6W5B) Rabbit mAb #14793 (lower).
Immunoprecipitation of BRD4 protein from 293T cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is BRD4 (pan) (E1Y1P) Rabbit mAb. Western blot analysis was performed using BRD4 (pan) (E1Y1P) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.
Chromatin immunoprecipitations were performed with cross-linked chromatin from MV-4-11 cells and either BRD4 (pan) (E1Y1P) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human c-Myc Intron 1 Primers #14905, SimpleChIP® Human Bcl-2 Promoter Primers #12924, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using BRD4 (E2A7X) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human large cell neuroendocrine carcinoma of the lung using BRD4 (E2A7X) Rabbit mAb (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).
Immunohistochemical analysis of paraffin-embedded human non-Hodgkin lymphoma using BRD4 (E2A7X) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded normal human pancreas using BRD4 (E2A7X) Rabbit mAb.
Chromatin immunoprecipitations were performed with cross-linked chromatin from MV-4-11 cells and BRD4 (E2A7X) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across MYC, a known target gene of BRD4 (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.
Chromatin immunoprecipitations were performed with cross-linked chromatin from MV-4-11 cells and BRD4 (E2A7X) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across chromosome 8 (upper), including MYC (lower), a known target gene of BRD4 (see additional figure containing ChIP-qPCR data).
Chromatin immunoprecipitations were performed with cross-linked chromatin from MV-4-11 cells and either BRD4 (E2A7X) Rabbit mAb or Normal Rabbit IgG #2729, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human Bcl-2 Promoter Primers #12924, human c-Myc intron 1 primers, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
CUT&RUN was performed with MV-4-11 cells and BRD4 (E2A7X) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across TEME53 gene, a known target gene of BRD4 (see additional figure containing CUT&RUN-qPCR data).
CUT&RUN was performed with MV-4-11 cells and BRD4 (E2A7X) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across LINC01132 gene (upper) and TEME53 gene (lower), a known target gene of BRD4 (see additional figure containing CUT&RUN-qPCR data).
CUT&RUN was performed with MV-4-11 cells and either BRD4 (E2A7X) Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using human TMEM53 exon 1 primers and human TBX15 downstream primers. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
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To Purchase # 82970T
Cat. # Size Price Inventory
82970T		 1 Kit  (3 x 20 microliters)

Bulk & Custom Formulation

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
BRD4 (E2A7X) Rabbit mAb 13440 20 µl
  • WB
  • IP
  • IHC
  • ChIP
  • C&R
  • eCLIP
 H 200 Rabbit IgG
BRD4 Isoform C (E1Q9N) Rabbit mAb 20696 20 µl
  • WB
  • IP
  • IF
  • ChIP
 H M R Mk 105 Rabbit IgG
BRD4 (pan) (E1Y1P) Rabbit mAb 83375 20 µl
  • WB
  • IP
  • ChIP
 H M R Mk 200, 110 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
 Goat 

Product Description

The BRD4 Antibody Sampler Kit provides an economical means of detecting various BRD4 isoforms. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

Background

Bromodomain-containing protein 4 (BRD4) is a member of the bromodomains and extra terminal (BET) family of proteins, which also includes BRD2, BRD3, and BRDT (1-3). BET family proteins contain two tandem bromodomains and an extra terminal (ET) domain, and bind acetyl lysine residues (3). BRD4 is a chromatin-binding protein with a preference for Lys14 on histone H3 as well as Lys5 and Lys12 on histone H4 (4). BRD4 chromatin binding occurs throughout the cell cycle, including condensed mitotic chromosomes, when the majority of genes are silenced (5). BRD4 association with chromatin during mitosis is thought to be an important part of the bookmarking mechanism to accelerate re-activation of the silenced genes upon exit from mitosis (2,6). BRD4 has been shown to facilitate transcription by recruiting the positive transcription elongation factor b (pTEFb) complex that phosphorylates Ser2 of the heptapeptide repeat of the carboxy-terminal domain of RNA polymerase II, promoting transcription elongation (3,7,8). In addition, BRD4 has been found to be part of the super elongation complex and the polymerase associated factor complex (PAFc) in MLL-fusion derived leukemia cell lines, demonstrating a role for BRD4 in the regulation of transcription elongation (9). Research studies have shown that BRD4 (and BET family proteins) may be promising therapeutic targets for various Myc-driven cancers, such as Burkitt’s lymphoma and certain acute myeloid leukemias (1,10,11). Investigators have found molecular inhibition of BET proteins to be effective in inducing apoptosis in various MLL-fusion driven leukemic cell lines by competing BRD3 and BRD4 from chromatin, leading to reduced expression of Bcl-2, Myc, and CDK6 (9). BET inhibition has also been shown to have antitumor activities against nuclear protein in testis (NUT) midline carcinoma cell lines and xenografts in mice where BRD4 is found to be a frequent translocation partner of the NUT protein (12). In addition, BRD4 regulates the expression of some inflammatory genes, and inhibition of BRD4 (and BET family proteins) chromatin binding causes reduced expression of a subset of inflammatory genes in macrophages, leading to protection against endotoxic shock and sepsis (13).

The expression of BRD4 isoform C, also known as the short isoform (BRD4-S), is similar to the long isoform (BRD4-L). The balance of expression between the two isoforms has been shown to contribute to disease states, where BRD4-S has shown to be oncogenic in a few contexts (14-16). BRD4-S is the predominant isoform that binds to modified histones and has a stronger affinity than BRD4-L (17,18). BRD4-S also plays a role in DNA damage, where it recruits the condensin II complex to enhance DNA damage induced cell death (18).

  1. Belkina, A.C. and Denis, G.V. (2012) Nat Rev Cancer 12, 465-77.
  2. Voigt, P. and Reinberg, D. (2011) Genome Biol 12, 133.
  3. Wu, S.Y. and Chiang, C.M. (2007) J Biol Chem 282, 13141-5.
  4. Dey, A. et al. (2003) Proc Natl Acad Sci U S A 100, 8758-63.
  5. Dey, A. et al. (2009) Mol Biol Cell 20, 4899-909.
  6. Zhao, R. et al. (2011) Nat Cell Biol 13, 1295-304.
  7. Jang, M.K. et al. (2005) Mol Cell 19, 523-34.
  8. Yang, Z. et al. (2005) Mol Cell 19, 535-45.
  9. Dawson, M.A. et al. (2011) Nature 478, 529-33.
  10. Muller, S. et al. (2011) Expert Rev Mol Med 13, e29.
  11. Mertz, J.A. et al. (2011) Proc Natl Acad Sci U S A 108, 16669-74.
  12. Filippakopoulos, P. et al. (2010) Nature 468, 1067-73.
  13. Nicodeme, E. et al. (2010) Nature 468, 1119-23.
  14. Wu, S.Y. et al. (2020) Mol Cell 78, 1114-1132.e10.
  15. Crawford, N.P. et al. (2008) Proc Natl Acad Sci USA 105, 6380-5.
  16. Fernandez, P. et al. (2014) Cell Rep 9, 248-260.
  17. Wang, R. et al. (2012) J Biol Chem 287, 10738-52.
  18. Han, X. et al. (2020) Nat Struct Mol Biol 27, 333-341.

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