AR CUTANA™ CUT&RUN Antibody

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. The androgen receptor (AR) is a transcription factor that is activated by steroid hormones. Upon binding, AR translocates into the nucleus and regulates the activity of androgen-responsive genes [1]. AR antibody produces CUT&RUN peaks above background (Figure 1) within gene promoters, intergenic, and intronic regions (Figures 1-2).

CUT&RUN was performed on 500k HeLa cells with 0.5 µg of either AR 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 5.1 million reads (IgG), 10.1 million reads (AR), and 6.2 million reads (H3K4me3). Data were aligned to the hg19 genome using Bowtie2. Data were filtered to remove duplicates, multi-aligned reads, and ENCODE DAC Exclusion List regions.

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Figure 1: AR peaks in CUT&RUN
CUT&RUN was performed as described above. Peaks were called with MACS2. Heatmaps show AR peaks relative to IgG negative 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 (left). The number of peaks that fall into distinct classes of functionally annotated genomic regions are shown (right).

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Figure 2: AR CUT&RUN representative browser tracks
CUT&RUN was performed as described above. Gene browser shots were generated using the Integrative Genomics Viewer (IGV, Broad Institute). Three gene loci show AR peaks at known androgen-responsive genes.

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Figure 3: Immunohistochemistry data
FFPE section of human ovarian carcinoma using AR antibody at a dilution of 1:1,000.

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

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Figure 5: Western blot data
Western analysis of AR in whole cell extracts from LNCaP (15 and 50 µg), HeLa (50 µg), and HEK293T (50 µg) cells. Lysates were resolved via SDS-PAGE and detected with a 1:2,500 dilution of AR antibody.