Systematic comparison of monoclonal versus polyclonal antibodies for mapping histone modifications by ChIP-seq.

Charles B. Epstein,Ido Yofe,Yossi Farjoun,Evan M. Cornett,Catherine Xue,Scott B. Rothbart,Michele Busby,Adrianne Gladden,Catherine Li,Elizabeth Gienger,Alon Goren,Chad Nusbaum

doi:10.1186/s13072-016-0100-6

Abstract

BackgroundThe robustness of ChIP-seq datasets is highly dependent upon the antibodies used. Currently, polyclonal antibodies are the standard despite several limitations: They are non-renewable, vary in performance between lots and need to be validated with each new lot. In contrast, monoclonal antibody lots are renewable and provide consistent performance. To increase ChIP-seq standardization, we investigated whether monoclonal antibodies could replace polyclonal antibodies. We compared monoclonal antibodies that target five key histone modifications (H3K4me1, H3K4me3, H3K9me3, H3K27ac and H3K27me3) to their polyclonal counterparts in both human and mouse cells.ResultsOverall performance was highly similar for four monoclonal/polyclonal pairs, including when we used two distinct lots of the same monoclonal antibody. In contrast, the binding patterns for H3K27ac differed substantially between polyclonal and monoclonal antibodies. However, this was most likely due to the distinct immunogen used rather than the clonality of the antibody.Conclusions Altogether, we found that monoclonal antibodies as a class perform equivalently to polyclonal antibodies for the detection of histone post-translational modifications in both human and mouse. Accordingly, we recommend the use of monoclonal antibodies in ChIP-seq experiments.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-016-0100-6) contains supplementary material, which is available to authorized users.

Highlights

The robustness of Chromatin immunoprecipitation (ChIP)-seq datasets is highly dependent upon the antibodies used
We found that the performance of monoclonal antibodies targeting histone post-translational modifications in ChIP-seq assays matched the performance of polyclonal antibodies
We designed an experimental system for rigorously comparing the performance of monoclonal and polyclonal antibodies in ChIP-seq and applied it to antibodies targeting five key histone modifications (H3K4me1, H3K4me3, H3K9me3, H3K27ac and H3K27me3) (Table 1)

Summary

Introduction

The robustness of ChIP-seq datasets is highly dependent upon the antibodies used. Monoclonal antibody lots are renewable and provide consistent performance. To increase ChIP-seq standardization, we investigated whether monoclonal antibodies could replace polyclonal antibodies. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is one of the key technologies for investigating the genomic localization of DNA-associated proteins. Busby et al Epigenetics & Chromatin (2016) 9:49 individual antibody molecules, representing the unique response of the source animal’s immune system. Some of these component antibody molecules will target the epitope in question, but other molecules in this population may enrich for other off-target epitopes. Once exhausted, a polyclonal antibody lot cannot be reproduced [6]

Methods

Results

Conclusion