BRG1/SMARCA4

BRG1/SMARCA4 CUTANA™ CUT&RUN Antibody

$495.00
SKU: 13-2002
Pack size: 100 µL
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  • Type:  Polyclonal
  • Host:  Rabbit
  • Applications: CUT&RUN, IHC, IP, WB
  • Reactivity:  Human, Mouse
  • Format:  Antigen affinity-purified
  • Target Size:  185 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 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. BRG1 antibody produces CUT&RUN peaks primarily flanking transcription start sites (TSSs, Figure 1). BRG1 peaks show a large degree of overlap with BRD4 peaks (Figure 2), as has been reported in the literature [Conrad et al., 2017].

Validation Data

CUT&RUN was performed on 500k K562 cells with 0.5 µg of either BRG1/SMARCA4, 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 8.7 million reads (BRG1), 4.1 million reads (H3K4me3), and 2.4 million reads (IgG) Data were aligned to the hg19 genome using Bowtie2. Data were filtered to remove duplicates, multi-aligned reads, and ENCODE DAC Exclusion List regions.

13_2002_Heatmap (Click to enlarge)

Figure 1: BRG1 peaks in CUT&RUN.
CUT&RUN was performed as described above. Peaks were called with MACS2. Heatmap showing BRG1 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.

13_2002_tracks (Click to enlarge)

Figure 2: BRG1 CUT&RUN peak enrichment and functional overlap.
CUT&RUN was performed as described above. BRG1 peaks overlapped with BRD4 antibody CUT&RUN peaks (EpiCypher 13-2003, top), as has been demonstrated in the literature [1]. Gene browser shots were generated using the Integrative Genomics Viewer (IGV, Broad Institute). Three representative loci show overlap of BRG1 peaks with H3K4me3 and BRD4 peaks (bottom).

13_2002_WB_H (Click to enlarge)

Figure 3: Immunohistochemistry data.
(A) FFPE section of human ovarian carcinoma using BRG1 antibody at a dilution of 1:1,000. (B) FFPE section of mouse renal cell carcinoma using BRG1 antibody at a dilution of 1:1,000.

13_2002_WB_M (Click to enlarge)

Figure 4: Western blot data.
Western analysis of BRG1 in whole cell lysates were isolated from HeLa, HEK293T, and Jurkat cells using NETN lysis buffer. Lysates (15 µg) were loaded onto 4-8% SDS-PAGE gel and analyzed under standard western blot conditions using BRG1 antibody (0.1 µg/mL).

13_2002_IP (Click to enlarge)

Figure 5: Western blot data.
Western analysis of BRG1 in whole cell lysates from NIH 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 BRG1 antibody (0.1 µg/mL).

13_2002_IHC (Click to enlarge)

Figure 6: Immunoprecipitation data.
EpiCypher BRG1 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 antibody to a different BRG1/SMARCA4 epitope (Bethyl Laboratories) were also used to demonstrate specificity of the IP. Immunoprecipitates were loaded onto 4-20% SDS-PAGE gel (20% of IP loaded) and probed via western blot with EpiCypher BRG1 antibody (1 µg/mL).

Technical Information

Immunogen
Between amino acids 75 and 125
Storage
Stable for 1 year at 4°C from date of receipt
Formulation
Antigen affinity-purified antibody in Tris-buffered saline, 0.1% BSA, 0.09% sodium azide

Recommended Dilution

CUT&RUN:
0.5 µg per reaction
Immunoprecipitation (IP):
2 - 5 µg/mg lysate
Immunohistochemistry (IHC):
1:250 - 1:2,000. Epitope retrieval with citrate buffer pH 6.0 is recommended for FFPE tissue sections
Western Blot (WB):
1:2,000 - 1:10,000

Gene & Protein Information

UniProt ID
P51532
Gene Name
SMARCA4
Protein Name
Transcription activator BRG1
Target Size
185 kDa
Alternate Names
BAF190A, BRG1, SNF2B, SNF2L4, ATP-dependent helicase SMARCA4, BRG1-associated factor 190A (BAF190A), Mitotic growth and transcription activator, Protein BRG-1, Protein brahma homolog 1

Documents & Resources

Technical Datasheet

Additional Info

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

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