Estrogen Receptor Alpha (N-Terminal) 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. Estrogen Receptor Alpha N-terminal (ER alpha N-term) antibody shows CUT&RUN peaks in response to estradiol stimulation (Figure 1) that overlap with known estrogen response element (ERE) binding motifs (Figure 2). Overlap is further observed with peaks from an antibody to a different ER alpha epitope (C-term) and NCOA3 (SRC3), which co-activates ER-mediated transcription [1] (Figure 2).

Serum-starved MCF7 cells were treated with estradiol (E2) or vehicle control for 45 minutes. CUT&RUN was performed on 500k cells with 0.5 µg of either ER Alpha (N-Term), ER Alpha (C-Term; EpiCypher 13-2012), NOCA3/SRC3 (EpiCypher 13-2013), 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). 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: ER alpha N-term peaks in CUT&RUN
CUT&RUN was performed as described above. Heatmaps show ER alpha N-term peaks relative to IgG negative control, H3K4me3 positive control, ER alpha C-term, and SRC3 antibodies in aligned rows ranked by intensity (top to bottom) and colored such that red indicates high localized enrichment and blue denotes background signal.

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Figure 2: ER alpha N-term peak analysis in CUT&RUN
Peaks from the E2-treated samples in Figure 1 were called using MACS2. (A) The number of ER alpha N-term peaks which fall into distinct classes of functionally annotated genomic regions is plotted. (B) Homer analysis determined that the ERE consensus motif, represented as a sequence logo position weight matrix, was enriched under ER alpha N-term peaks. (C) The number of ER alpha N-term peaks containing consensus motifs from panel B is shown by Venn Diagram. (D) The number of ER alpha N-term peaks that overlap with ER alpha C-term and SRC3 antibodies are represented by Venn Diagram.

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Figure 3: Immunoprecipitation data
EpiCypher ER alpha N-term antibody (3 µg) was used to immunoprecipitate whole cell lysates isolated from MCF7 cells (1.0 mg per IP). A negative control IgG antibody and positive control antibody to different ER alpha epitopes (EpiCypher 13-2012 and Bethyl Laboratories) were also used to demonstrate specificity of the IP. Immunoprecipitates were loaded onto a 4-8% SDS-PAGE gel (25% of IP loaded) and probed via western blot with EpiCypher 13-2012 ER alpha C-term antibody (0.1 µg/mL).

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Figure 4: Immunohistochemistry data
FFPE section of human breast examined using ER alpha N-term antibody (1:2,500 dilution, 0.4 µg/mL).