Optimized protocols for chromatin immunoprecipitation of exogenously expressed epitope-tagged proteins.
Optimized protocols for chromatin immunoprecipitation of exogenously expressed epitope-tagged proteins.
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DOI：
10.1016/j.xpro.2023.102050
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发表时间：
2023-03-17
期刊：
影响因子：
--
通讯作者：
Zhang, Qing
中科院分区：
文献类型：
--
作者：
Fang, Wentong;Liao, Chengheng;Zhang, Qing
染色质免疫沉淀（ChIP）分析广泛用于研究DNA与DNA结合蛋白（如转录因子、辅因子或染色质相关蛋白）之间的相互作用。然而，成功的ChIP检测在很大程度上取决于ChIP级一抗的质量。在特异性抗体不可用或结合亲和力低的情况下，在这里，我们描述了一种定制的方案，通过在细胞中表达外源表位标记的蛋白质，然后使用标签特异性抗体进行ChIP测定，来实现稳健和可重现的染色质结合。有关本方案使用和执行的完整详细信息，请参见Fang et al.（2021）和Kidder et al.（2011年）。表达外源性蛋白质并耗尽细胞中天然对应物的方案当无法获得蛋白质的ChIP级抗体时，另一种ChIP策略通过使用标签特异性抗体优化ChIP方案提供了优化的ChIP条件出版商注释：进行任何实验方案都需要遵守当地机构的实验室安全和伦理指南。染色质免疫沉淀（ChIP）分析广泛用于研究DNA与DNA结合蛋白（如转录因子、辅因子或染色质相关蛋白）之间的相互作用。然而，成功的ChIP检测在很大程度上取决于ChIP级一抗的质量。在特异性抗体不可用或结合亲和力低的情况下，在这里，我们描述了一种定制的方案，通过在细胞中表达外源表位标记的蛋白质，然后使用标签特异性抗体进行ChIP测定，来实现稳健和可重现的染色质结合。
Chromatin immunoprecipitation (ChIP) assay is widely used for investigating the interaction between DNA and DNA-binding proteins such as transcription factors, co-factors, or chromatin-associated proteins. However, a successful ChIP assay largely depends on the quality of a ChIP-grade primary antibody. In cases where specific antibodies are unavailable or with low binding affinity, here, we describe a tailored protocol to achieve robust and reproducible chromatin binding by expressing an exogenous epitope-tagged protein in cells, followed by ChIP assays using a tag-specific antibody. For complete details on the use and execution of this protocol, please refer to Fang et al. (2021) and Kidder et al. (2011). Protocol to express an exogenous protein and depleting its native counterpart in cells An alternative ChIP strategy when ChIP-grade antibody of a protein is unavailable Optimized ChIP protocol by using a tag-specific antibody Optimized ChIP conditions were provided Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Chromatin immunoprecipitation (ChIP) assay is widely used for investigating the interaction between DNA and DNA-binding proteins such as transcription factors, co-factors, or chromatin-associated proteins. However, a successful ChIP assay largely depends on the quality of a ChIP-grade primary antibody. In cases where specific antibodies are unavailable or with low binding affinity, here, we describe a tailored protocol to achieve robust and reproducible chromatin binding by expressing an exogenous epitope-tagged protein in cells, followed by ChIP assays using a tag-specific antibody.
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影响因子：
4
作者：
Jiang J;Sun Y;Xu J;Xu T;Xu Z;Liu P
通讯作者：
Liu P
影响因子：
5.8
作者：
Zhang Y;Sun M;Gao L;Liang X;Ma C;Lu J;Yue X
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Yue X
影响因子：
--
作者：
Reece-Hoyes JS;Walhout AJM
通讯作者：
Walhout AJM
DOI：
10.1126/science.aap8411
发表时间：
2018-07-20
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Zhang J;Wu T;Simon J;Takada M;Saito R;Fan C;Liu XD;Jonasch E;Xie L;Chen X;Yao X;Teh BT;Tan P;Zheng X;Li M;Lawrence C;Fan J;Geng J;Liu X;Hu L;Wang J;Liao C;Hong K;Zurlo G;Parker JS;Auman JT;Perou CM;Rathmell WK;Kim WY;Kirschner MW;Kaelin WG Jr;Baldwin AS;Zhang Q
通讯作者：
Zhang Q
影响因子：
30.5
作者：
Kidder, Benjamin L.;Hu, Gangqing;Zhao, Keji
通讯作者：
Zhao, Keji