BRD4

BRD4 CUTANA™ CUT&RUN Antibody

$445.00
SKU: 13-2003
Pack size: 50 µL
  • Type:  Polyclonal
  • Host:  Rabbit
  • Target Size:  152 kDa
  • Format:  Affinity Purified IgG
  • Reactivity:  Human, Mouse
  • Applications: CUT&RUN, WB, IP, IHC

Description

This antibody meets EpiCypher’s “CUTANA Compatible” criteria for performance in Cleavage Under Targets and Release Using Nuclease (CUT&RUN) and/or Cleavage Under Targets and Tagmentation (CUT&Tag) approaches to genomic mapping. Every lot of a CUTANA Compatible antibody is tested in the indicated CUTANA approach using EpiCypher optimized protocols and determined to yield peaks that show a genomic distribution pattern consistent with reported function(s) of the target protein. BRD4 antibody produces CUT&RUN peaks primarily flanking transcription start sites (TSSs, Figure 1). BRD4 peaks show a large degree of overlap with BRG1/SMARCA4 peaks (Figure 2), as has been reported in the literature (Conrad et al., 2017).

Validation Data

Figure 1: BRD4 enrichment at annotated TSSs in CUT&RUN
CUT&RUN was performed using 500,000 K562 cells with BRD4 antibody as well as control antibodies (IgG negative control, EpiCypher 13-0042; H3K4me3 positive control, EpiCypher 13-0041). Sequencing reads were aligned to annotated TSSs (+/- 2 kbp) of 18,793 genes. High, medium, and low signal is ranked by intensity (top to bottom) and reflected by red, yellow, and blue colors, respectively. All rows aligned relative to H3K4me3 antibody.

Figure 2: BRD4 CUT&RUN peak enrichment and functional overlap
The CUT&RUN data from Figure 1 was subjected to peak calling using MACS2. BRD4 peaks overlapped with BRG1/ SMARCA4 antibody CUT&RUN peaks (EpiCypher 13-2002, top), as has been demonstrated in the literature (Conrad et al., 2017). Three representative loci show BRD4 peaks in relation to control and BRG1 antibodies (bottom, Integrative Genomics Viewer).

Figure 3: Western blot detection of human BRD4
Whole cell lysates were isolated from HeLa, HEK293T (“293T”), Jurkat, MB231, Hep-G2, A-549, SW620, SK-MEL-28 (“SK”), and Ramos cells using NETN lysis buffer. Lysates (15 µg) were loaded onto 4-8% SDS-PAGE gel and analyzed under standard western blot conditions using BRD4 antibody (1:1,000 dilution).

Figure 4: Western blot detection of mouse BRD4
Whole cell lysates were isolated from NIH 3T3 (“3T3”) cells using NETN lysis buffer. Lysates (15 µg) were loaded onto 4-8% SDS-PAGE gel and analyzed under standard western blot conditions using BRD4 antibody (0.1 µg/mL).

Figure 5: Immunoprecipitation of human BRD4
EpiCypher BRD4 antibody (3 µg) was used to immunoprecipitate whole cell lysates isolated from HeLa cells using NETN lysis buffer (1 mg per IP). A negative control IgG antibody and positive control antibodies to a different BRD4 epitopes (Bethyl Laboratories) were also used to demonstrate specificity of the IP. Immunoprecipitates were loaded onto 4-8% SDS-PAGE gel (20% of IP loaded) and probed via western blot with a different BRD4 antibody (Bethyl Laboratories, Catalog No. A700-004, 1:1,000).

Figure 6: Immunohistochemistry detection of human and mouse BRD4
(A) FFPE section of human ovarian carcinoma examined using BRD4 antibody (1:5,000 dilution, 0.2 µg/mL). (B) FFPE section of mouse CT26 colon carcinoma examined using BRD4 antibody (1:1,000 dilution, 1 µg/mL).

Technical Information

Immunogen
A synthetic peptide corresponding to human BRD4 amino acids 1312 to 1362.
Formulation
Antigen affinity-purified antibody (1 mg/mL) in Tris-citrate/phosphate buffer pH 7 to 8, 0.09% sodium azide.
Storage and Stability
Stable for 1 year at 4°C from date of receipt.

Application Notes

Recommended Dilutions:

CUT&RUN: 0.5 µg

Western Blot (WB): 1:2,000 - 1:10,000

Immunoprecipitation (IP): 2 - 5 µg/mg lysate

Immunohistochemistry (IHC): 1:1,000 - 1:5,000*

*Epitope retrieval with citrate buffer pH 6.0 recommended for FFPE tissue

References

Background References:
Conrad et al. Mol. Cell (2017). PMID: 28844864

Documents & Resources

Additional Info

This product is provided for commercial sale under license from Bethyl Laboratories, Inc.

Applications Key:

ChIP: Chromatin immunoprecipitation
CUT&RUN: Cleavage Under Targets and Release Using Nuclease
CUT&Tag: Cleavage Under Targets and Tagmentation
E: ELISA
FACS: Flow cytometry
ICC: Immunocytochemistry
IF: Immunofluorescence
IHC: Immunohistochemistry
IP: Immunoprecipitation
L: Luminex
WB: Western Blot

Reactivity Key:

B: Bovine
Ce: C. elegans
Ch: Chicken
Dm: Drosophila
Eu: Eukaryote
H: Human
M: Mouse
Ma: Mammal
R: Rat
Sc: S. cerevisiae
Sp: S. pombe
WR: Wide Range (predicted)
X: Xenopus
Z: Zebrafish
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