BRD4

BRD4 CUTANA™ CUT&RUN Antibody

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

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 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 [1].

Validation Data

CUT&RUN was performed on 500k K562 cells with 0.5 µg of either BRD4, BRG1, H3K4me3 positive control (EpiCypher 13-0041), or IgG negative control (EpiCypher 13-0042) antibodies using the CUTANA™ ChIC/CUT&RUN Kit v2.0 (EpiCypher 14-1048). Library preparation was performed with 5 ng of DNA (or the total amount recovered if less than 5 ng) using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001/14-1002). Both kit protocols were adapted for high throughput Tecan liquid handling. Libraries were run on an Illumina NextSeq2000 with paired-end sequencing (2x50 bp). Sample sequencing depth was 9.4 million reads (BRD4), 7.5 million reads (BRG1), 11.7 million reads (H3K4me3), and 8.3 million reads (IgG). Data were aligned to the hg19 genome using Bowtie2. Data were filtered to remove duplicates, multi-aligned reads, and blacklist regions.

13-2003_Peaks (Click to enlarge)

Figure 1: BRD4 peaks in CUT&RUN
CUT&RUN was performed as described above. Peaks were called with MACS2. Heatmaps show BRD4 peaks relative to IgG negative control antibody and H3K4me3 positive control antibody in aligned rows ranked by intensity (top to bottom) and colored such that red indicates high localized enrichment and blue denotes background signal. All rows were aligned relative to H3K4me3 antibody.

13-2003_Tracks (Click to enlarge)

Figure 2: BRD4 CUT&RUN representative browser tracks
CUT&RUN was performed as described above. Gene browser shots were generated using the Integrate Genomics Viewer (IGV, Broad Institute). Three representative loci show overlap of BRD4 and BRG1 peaks.

13-2003_IHC (Click to enlarge)

Figure 3: Immunohistochemistry data
FFPE sections of human ovarian carcinoma (left) and mouse renal cell carcinoma (right) using BRD4 antibody at a dilution of 1:5,000.

13-2003_IP (Click to enlarge)

Figure 4: Immunoprecipitation data
EpiCypher BRD4 antibody (6 µg) was used to immunoprecipitate whole cell lysates (1 mg, 20% of IP loaded) isolated from HeLa cells. A negative control IgG antibody and positive control antibody targeting BRD4 (Bethyl Laboratories) were also used to demonstrate the specificity of the IP. For blotting immunoprecipitates, Bethyl Laboratories BRD4 antibody (A700-004) was used at a dilution of 1:1,000.

13-2003_WB_HeLa (Click to enlarge)

Figure 5: Western blot data
Western analysis of BRD4 in whole cell extracts from HeLa, HEK293T, MDA-MB-231, Hep-G2, A-549, SW620, and SK-MEL-28 cells. Ten micrograms of lysate was resolved via SDS-page and detected with a 1:25,000 dilution of EpiCypher BRD4 antibody.

13-2003_WB_BW5147.3 (Click to enlarge)

Figure 6: Western blot data
Western analysis of BRD4 in whole cell extracts from BW5147.3, ELF4, RenCa, NIH3T3, and TCMK-1 cells. Fifteen micrograms of lysate was resolved via SDS-page and detected with a 1:25,000 dilution of EpiCypher BRD4 antibody.

Technical Information

Immunogen
Between amino acids 1312 and 1362
Storage and Stability
Stable for 1 year at 4°C from date of receipt
Formulation
Antigen affinity-purified antibody in Tris-citrate/phosphate buffer pH 7-8, 0.09% sodium azide

Recommended Dilution

CUT&RUN:
0.5 µg per reaction
Immunohistochemistry (IHC):
1:1,000 - 1:5,000. Epitope retrieval with citrate buffer pH 6.0 is recommended for FFPE tissue sections
Immunoprecipitation (IP):
2 - 5 µg/mg lysate
Western Blot (WB):
1:2,000 - 1:10,000

Gene & Protein Information

UniProt ID
O60885
Gene Name
BRD4
Protein Name
Bromodomain-containing protein 4
Target Size
152 kDa
Alternate Names
HUNK1, Protein HUNK1, bromodomain-containing protein 4

References

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

Documents & Resources

Technical Datasheet

Additional Info

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

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