A ChIP-exo screen of 887 Protein Capture Reagents Program transcription factor antibodies in human cells.
A ChIP-exo screen of 887 Protein Capture Reagents Program transcription factor antibodies in human cells.
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DOI：
10.1101/gr.275472.121
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发表时间：
2021-09
期刊：
影响因子：
7
通讯作者：
Pugh BF
中科院分区：
文献类型：
--
作者：
Lai WKM;Mariani L;Rothschild G;Smith ER;Venters BJ;Blanda TR;Kuntala PK;Bocklund K;Mairose J;Dweikat SN;Mistretta K;Rossi MJ;James D;Anderson JT;Phanor SK;Zhang W;Zhao Z;Shah AP;Novitzky K;McAnarney E;Keogh MC;Shilatifard A;Basu U;Bulyk ML;Pugh BF
抗体提供了一种强有力的手段来询问复杂环境中的特定蛋白质。然而，抗体的可用性和可靠性可能是有问题的，而表位标记在许多情况下可能是不切实际的。为了解决这些局限性，蛋白质捕获试剂计划（PCRP）产生了超过一千种针对人类假定染色质蛋白的可再生单克隆抗体（mAb）。然而，这些试剂尚未经过广泛的实地测试。因此，我们进行了筛选，以测试他们的能力，丰富的基因组区域通过染色质免疫沉淀（ChIP）和各种正交试验。通过超高分辨率ChIP-exo/seq分析了针对681种独特人转录因子（TF）的887种独特抗体，生成约1200个ChIP-exo数据集，主要在一种细胞类型（K562）中进行单次通过。在ChIP-seq、CUT&RUN、STORM超分辨率显微镜、免疫印迹和蛋白质结合微阵列（PBM）实验中进一步测试PCRP mAb的亚组。约5%的测试抗体显示出高置信度靶标（即，同源抗原）富集，并且是用于额外验证的强候选者。另外34%产生的ChIP-exo数据与背景不同，因此需要进一步测试。其余61%与背景无显著差异，可能需要考虑更广泛的细胞类型调查和/或测定优化。我们展示并讨论了基于染色质的检测中抗体验证的指标和挑战。
Antibodies offer a powerful means to interrogate specific proteins in a complex milieu. However, antibody availability and reliability can be problematic, whereas epitope tagging can be impractical in many cases. To address these limitations, the Protein Capture Reagents Program (PCRP) generated over a thousand renewable monoclonal antibodies (mAbs) against human presumptive chromatin proteins. However, these reagents have not been widely field-tested. We therefore performed a screen to test their ability to enrich genomic regions via chromatin immunoprecipitation (ChIP) and a variety of orthogonal assays. Eight hundred eighty-seven unique antibodies against 681 unique human transcription factors (TFs) were assayed by ultra-high-resolution ChIP-exo/seq, generating approximately 1200 ChIP-exo data sets, primarily in a single pass in one cell type (K562). Subsets of PCRP mAbs were further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and protein binding microarray (PBM) experiments. About 5% of the tested antibodies displayed high-confidence target (i.e., cognate antigen) enrichment across at least one assay and are strong candidates for additional validation. An additional 34% produced ChIP-exo data that were distinct from background and thus warrant further testing. The remaining 61% were not substantially different from background, and likely require consideration of a much broader survey of cell types and/or assay optimizations. We show and discuss the metrics and challenges to antibody validation in chromatin-based assays.
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影响因子：
64.8
作者：
Ernst, Jason;Kheradpour, Pouya;Mikkelsen, Tarjei S.;Shoresh, Noam;Ward, Lucas D.;Epstein, Charles B.;Zhang, Xiaolan;Wang, Li;Issner, Robbyn;Coyne, Michael;Ku, Manching;Durham, Timothy;Kellis, Manolis;Bernstein, Bradley E.
通讯作者：
Bernstein, Bradley E.
影响因子：
14.8
作者：
Berger, Michael F.;Bulyk, Martha L.
通讯作者：
Bulyk, Martha L.
影响因子：
46.9
作者：
Berger, Michael F.;Philippakis, Anthony A.;Bulyk, Martha L.
通讯作者：
Bulyk, Martha L.
影响因子：
7
作者：
Gordan, Raluca;Hartemink, Alexander J.;Bulyk, Martha L.
通讯作者：
Bulyk, Martha L.
影响因子：
5.3
作者：
BALER, R;DAHL, G;VOELLMY, R
通讯作者：
VOELLMY, R