Broad ranges of affinity and specificity of anti-histone antibodies revealed by a quantitative peptide immunoprecipitation assay.
Broad ranges of affinity and specificity of anti-histone antibodies revealed by a quantitative peptide immunoprecipitation assay.
复制标题
DOI：
10.1016/j.jmb.2012.09.022
复制
发表时间：
2012-12-14
影响因子：
5.6
通讯作者：
Koide, Shohei
中科院分区：
文献类型：
--
作者：
Nishikori, Shingo;Hattori, Takamitsu;Fuchs, Stephen M.;Yasui, Norihisa;Wojcik, John;Koide, Akiko;Strahl, Brian D.;Koide, Shohei
针对组蛋白翻译后修饰（PTMs）的抗体是表观遗传学研究的重要工具，特别是在广泛使用的染色质免疫沉淀（ChIP）实验中。然而，缺乏表征这些抗体的定量方法一直是准确和可重复分析组蛋白修饰的主要瓶颈。在这里，我们报告了一种简单而敏感的方法，用于在芯片样格式中定量表征组蛋白PTMs的多克隆和单克隆抗体。重要的是，它确定了抗体与含有同源或脱靶PTMs的肽相互作用的表观解离常数。对商业抗体的分析显示，这些抗体具有很大的亲和力、特异性和结合能力，以及大量的批次间差异，这表明定量表征每种拟用于ChIP实验的抗体并相应地优化实验条件的重要性。此外，使用这种方法，我们确定了可能影响ChIP实验解释的其他因素。
Antibodies directed against histone posttranslational modifications (PTMs) are critical tools in epigenetics research, particularly in the widely used chromatin immunoprecipitation (ChIP) experiments. However, a lack of quantitative methods for characterizing such antibodies has been a major bottleneck in accurate and reproducible analysis of histone modifications. Here, we report a simple and sensitive method for quantitatively characterizing polyclonal and monoclonal antibodies for histone PTMs in a ChIP-like format. Importantly, it determines the apparent dissociation constants for the interactions of an antibody with peptides harboring cognate or off-target PTMs. Analyses of commercial antibodies revealed large ranges of affinity, specificity and binding capacity as well as substantial lot-to-lot variations, suggesting the importance of quantitatively characterizing each antibody intended to be used in ChIP experiments and optimizing experimental conditions accordingly. Furthermore, using this method we identified additional factors potentially affecting the interpretation of ChIP experiments.
登录
查看更多内容
影响因子：
14.8
作者：
Brand, Marjorie;Rampalli, Shravanti;Dilworth, F. Jeffrey
通讯作者：
Dilworth, F. Jeffrey
影响因子：
7
作者：
Peach, Sally E.;Rudomin, Emily L.;Jaffe, Jacob D.
通讯作者：
Jaffe, Jacob D.
影响因子：
9.2
作者：
Fuchs, Stephen M.;Krajewski, Krzysztof;Baker, Richard W.;Miller, Victoria L.;Strahl, Brian D.
通讯作者：
Strahl, Brian D.
组蛋白修饰抗体质量的评估。
影响因子：
16.8
作者：
通讯作者：
--
影响因子：
14.8
作者：
Chao, Ginger;Lau, Wai L.;Wittrup, K. Dane
通讯作者：
Wittrup, K. Dane