Research Article: Identification of ChIP-seq and RIME grade antibodies for Estrogen Receptor alpha

Date Published: April 10, 2019

Publisher: Public Library of Science

Author(s): Silvia-E. Glont, Evangelia K. Papachristou, Ashley Sawle, Kelly A. Holmes, Jason S. Carroll, Rasmus Siersbaek, Alessandro Weisz.

http://doi.org/10.1371/journal.pone.0215340

Abstract

Estrogen Receptor alpha (ERα) plays a major role in most breast cancers, and it is the target of endocrine therapies used in the clinic as standard of care for women with breast cancer expressing this receptor. The two methods ChIP-seq (chromatin immunoprecipitation coupled with deep sequencing) and RIME (Rapid Immunoprecipitation of Endogenous Proteins) have greatly improved our understanding of ERα function during breast cancer progression and in response to anti-estrogens. A critical component of both ChIP-seq and RIME protocols is the antibody that is used against the bait protein. To date, most of the ChIP-seq and RIME experiments for the study of ERα have been performed using the sc-543 antibody from Santa Cruz Biotechnology. However, this antibody has been discontinued, thereby severely impacting the study of ERα in normal physiology as well as diseases such as breast cancer and ovarian cancer. Here, we compare the sc-543 antibody with other commercially available antibodies, and we show that 06–935 (EMD Millipore) and ab3575 (Abcam) antibodies can successfully replace the sc-543 antibody for ChIP-seq and RIME experiments.

Partial Text

In the last decades, there has been significant interest in studying Estrogen Receptor alpha (ERα) due to its causal role in more than three quarters of breast cancers[1]. Its key role in breast cancer progression makes ERα the major target for endocrine therapies, which have substantially improved patient survival. However, resistance to these therapies occurs in many patients[2], which leads to incurable metastatic disease. Therefore, it is important to understand the mechanisms underlying ERα action in cancer initiation as well as progression of the disease. In addition, ERα plays an important role in development[3] and other diseases such as ovarian cancer[4].

Genome-wide analyses of ERα-chromatin binding sites using ChIP-based methods have exponentially increased our knowledge of the role of ERα in breast cancer. Most of the published ChIP-seq and RIME studies for ERα have been performed using the sc-543 antibody from Santa Cruz Biotechnology[13, 16, 17, 19–21, 32] and the quality and specificity of sc-543 has made it the ‘golden standard’ for immunoprecipitation experiments. However, this antibody has recently been discontinued, which has significantly impacted our ability to study ERα biology. Here, we have assessed commercially available alternative antibodies. We demonstrate using ChIP-seq and RIME that the two antibodies 06–935 (Millipore) and ab3575 (Abcam) perform similarly to sc-543, in terms of sensitivity and specificity. We therefore propose that these antibodies can replace the sc-543 antibody for immunoprecipitation-based experiments such as ChIP-seq and RIME to explore ERα function.

 

Source:

http://doi.org/10.1371/journal.pone.0215340

 

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