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Epigenetics Chromatin. 2016 Nov 4;9:49. eCollection 2016.

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

Author information

1
Broad Technology Labs, Broad Institute of MIT and Harvard, Cambridge, MA 02142 USA.
2
Broad Institute of MIT and Harvard, Cambridge, MA 02142 USA.
3
Broad Technology Labs, Broad Institute of MIT and Harvard, Cambridge, MA 02142 USA ; Department of Immunology, Weizmann Institute, 76100 Rehovot, Israel.
4
Center for Epigenetics, Van Andel Research Institute, Grand Rapids, MI 49503 USA.
5
Broad Technology Labs, Broad Institute of MIT and Harvard, Cambridge, MA 02142 USA ; Department of Medicine, University of California San Diego, La Jolla, CA 92093 USA.

Abstract

BACKGROUND:

The 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.

RESULTS:

Overall 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.

KEYWORDS:

Antibodies; ChIP-seq; Methods; Monoclonal; Polyclonal

PMID:
27826357
PMCID:
PMC5097419
DOI:
10.1186/s13072-016-0100-6
[Indexed for MEDLINE]
Free PMC Article

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