Estrogen

Estrogen Receptor Alpha (N-Terminal) CUTANA™ CUT&RUN Antibody

$445.00
SKU: 13-2011
Pack size: 100 µL
  • Type:  Polyclonal
  • Host:  Rabbit
  • Target Size:  66 kDa
  • Format:  Aff. Pur. IgG
  • Reactivity:  Human (Predicted: Mouse, Rat)
  • Applications: CUT&RUN, 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. 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; 13-2012) and NCOA3/SRC3 (13-2013), which coactivates ER-mediated transcription (1) (Figure 2).

Validation Data

Figure 1: ER alpha N-term enrichment in CUT&RUN
Serum-starved MCF7 cells were treated with 100 nM estradiol (E2) or vehicle control for 45 minutes. CUT&RUN was performed using 500,000 cells with 0.5 µg of each indicated antibody (gray text). Heatmap shows CUT&RUN enrichment in aligned rows ranked by intensity (top to bottom; relative to ER alpha C-term, 13-2012). Red indicates high localized enrichment and blue denotes background signal.

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 (13-2012) and NCOA3/SRC3 (13-2013) antibodies are represented by Venn Diagram.

Figure 3: Immunoprecipitation of human ER alpha
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 a different ER alpha epitopes (EpiCypher 13-2012 and Bethyl Laboratories) were also used to demonstrate specificity of the IP. Immunoprecipitates were loaded onto 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).

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

Technical Information

Immunogen
A synthetic peptide corresponding to human ER alpha amino acids 1-50.
Formulation
Antigen affinity-purified antibody (1.0 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

IHC: 1:1000 - 1:5,000*

IP: 3 - 5 µg/mg lysate

*Epitope retrieval with Tris-EDTA pH 9.0 recommended for FFPE tissue

References

Background References:
1. Wagner et al. BMC Cancer (2013). PMID: 24304549

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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