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A ChIP-exo screen of 887 Protein Capture Reagents Program transcription factor antibodies in human cells
Genome Research ( IF 7 ) Pub Date : 2021-09-01 , DOI: 10.1101/gr.275472.121
William K M Lai 1, 2 , Luca Mariani 3 , Gerson Rothschild 4 , Edwin R Smith 5 , Bryan J Venters 6 , Thomas R Blanda 1 , Prashant K Kuntala 1 , Kylie Bocklund 1 , Joshua Mairose 1 , Sarah N Dweikat 1 , Katelyn Mistretta 1 , Matthew J Rossi 1 , Daniela James 1 , James T Anderson 3 , Sabrina K Phanor 3 , Wanwei Zhang 4 , Zibo Zhao 5 , Avani P Shah 5 , Katherine Novitzky 6 , Eileen McAnarney 6 , Michael-C Keogh 6 , Ali Shilatifard 5 , Uttiya Basu 4 , Martha L Bulyk 3, 7 , B Franklin Pugh 1, 2
Affiliation  

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.

中文翻译:

887 Protein Capture Reagents Program 转录因子抗体在人类细胞中的 ChIP-exo 筛选

抗体提供了一种强大的手段来研究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存在问题,而表位标记在许多情况下可能是不切实际的。为了解决这些限制,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现场测试。因此,我们进行了筛选,以测试它们通过染色质免疫沉淀 (ChIP) 和各种正交测定来丰富基因组区域的能力。通过超高分辨率 ChIP-exo/seq 分析了针对 681 种独特人类转录因子 (TF) 的 887 种独特抗体,生成了大约 1200 个 ChIP-exo 数据集,主要在一种细胞类型 (K562) 中单次通过。PCRP mAb 的子集在 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) 实验中进行了进一步测试。大约 5% 的测试抗体在至少一种测定中显示出高置信度的靶标(即同源抗原)富集,并且是进一步验证的有力候选者。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% 与背景没有显着差异,可能需要考虑对细胞类型和/或分析优化进行更广泛的调查。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。STORM 超分辨率显微镜、免疫印迹和蛋白质结合微阵列 (PBM) 实验。大约 5% 的测试抗体在至少一种测定中显示出高置信度的靶标(即同源抗原)富集,并且是进一步验证的有力候选者。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% 与背景没有显着差异,可能需要考虑对细胞类型和/或分析优化进行更广泛的调查。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。STORM 超分辨率显微镜、免疫印迹和蛋白质结合微阵列 (PBM) 实验。大约 5% 的测试抗体在至少一种测定中显示出高置信度的靶标(即同源抗原)富集,并且是进一步验证的有力候选者。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% 与背景没有显着差异,可能需要考虑对细胞类型和/或分析优化进行更广泛的调查。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。同源抗原)在至少一种测定中的富集,并且是额外验证的有力候选者。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% 与背景没有显着差异,可能需要考虑对细胞类型和/或分析优化进行更广泛的调查。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。同源抗原)在至少一种测定中的富集,并且是额外验证的有力候选者。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% 与背景没有显着差异,可能需要考虑对细胞类型和/或分析优化进行更广泛的调查。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。
更新日期:2021-09-01
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