Cargando…
Systematic comparison of monoclonal versus polyclonal antibodies for mapping histone modifications by ChIP-seq
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 an...
Autores principales: | , , , , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2016
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5097419/ https://www.ncbi.nlm.nih.gov/pubmed/27826357 http://dx.doi.org/10.1186/s13072-016-0100-6 |
_version_ | 1782465598348853248 |
---|---|
author | Busby, Michele Xue, Catherine Li, Catherine Farjoun, Yossi Gienger, Elizabeth Yofe, Ido Gladden, Adrianne Epstein, Charles B. Cornett, Evan M. Rothbart, Scott B. Nusbaum, Chad Goren, Alon |
author_facet | Busby, Michele Xue, Catherine Li, Catherine Farjoun, Yossi Gienger, Elizabeth Yofe, Ido Gladden, Adrianne Epstein, Charles B. Cornett, Evan M. Rothbart, Scott B. Nusbaum, Chad Goren, Alon |
author_sort | Busby, Michele |
collection | PubMed |
description | 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13072-016-0100-6) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-5097419 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-50974192016-11-08 Systematic comparison of monoclonal versus polyclonal antibodies for mapping histone modifications by ChIP-seq Busby, Michele Xue, Catherine Li, Catherine Farjoun, Yossi Gienger, Elizabeth Yofe, Ido Gladden, Adrianne Epstein, Charles B. Cornett, Evan M. Rothbart, Scott B. Nusbaum, Chad Goren, Alon Epigenetics Chromatin Methodology 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13072-016-0100-6) contains supplementary material, which is available to authorized users. BioMed Central 2016-11-04 /pmc/articles/PMC5097419/ /pubmed/27826357 http://dx.doi.org/10.1186/s13072-016-0100-6 Text en © The Author(s) 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Methodology Busby, Michele Xue, Catherine Li, Catherine Farjoun, Yossi Gienger, Elizabeth Yofe, Ido Gladden, Adrianne Epstein, Charles B. Cornett, Evan M. Rothbart, Scott B. Nusbaum, Chad Goren, Alon Systematic comparison of monoclonal versus polyclonal antibodies for mapping histone modifications by ChIP-seq |
title | Systematic comparison of monoclonal versus polyclonal antibodies for mapping histone modifications by ChIP-seq |
title_full | Systematic comparison of monoclonal versus polyclonal antibodies for mapping histone modifications by ChIP-seq |
title_fullStr | Systematic comparison of monoclonal versus polyclonal antibodies for mapping histone modifications by ChIP-seq |
title_full_unstemmed | Systematic comparison of monoclonal versus polyclonal antibodies for mapping histone modifications by ChIP-seq |
title_short | Systematic comparison of monoclonal versus polyclonal antibodies for mapping histone modifications by ChIP-seq |
title_sort | systematic comparison of monoclonal versus polyclonal antibodies for mapping histone modifications by chip-seq |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5097419/ https://www.ncbi.nlm.nih.gov/pubmed/27826357 http://dx.doi.org/10.1186/s13072-016-0100-6 |
work_keys_str_mv | AT busbymichele systematiccomparisonofmonoclonalversuspolyclonalantibodiesformappinghistonemodificationsbychipseq AT xuecatherine systematiccomparisonofmonoclonalversuspolyclonalantibodiesformappinghistonemodificationsbychipseq AT licatherine systematiccomparisonofmonoclonalversuspolyclonalantibodiesformappinghistonemodificationsbychipseq AT farjounyossi systematiccomparisonofmonoclonalversuspolyclonalantibodiesformappinghistonemodificationsbychipseq AT giengerelizabeth systematiccomparisonofmonoclonalversuspolyclonalantibodiesformappinghistonemodificationsbychipseq AT yofeido systematiccomparisonofmonoclonalversuspolyclonalantibodiesformappinghistonemodificationsbychipseq AT gladdenadrianne systematiccomparisonofmonoclonalversuspolyclonalantibodiesformappinghistonemodificationsbychipseq AT epsteincharlesb systematiccomparisonofmonoclonalversuspolyclonalantibodiesformappinghistonemodificationsbychipseq AT cornettevanm systematiccomparisonofmonoclonalversuspolyclonalantibodiesformappinghistonemodificationsbychipseq AT rothbartscottb systematiccomparisonofmonoclonalversuspolyclonalantibodiesformappinghistonemodificationsbychipseq AT nusbaumchad systematiccomparisonofmonoclonalversuspolyclonalantibodiesformappinghistonemodificationsbychipseq AT gorenalon systematiccomparisonofmonoclonalversuspolyclonalantibodiesformappinghistonemodificationsbychipseq |