Antibody Validation in Bioimaging Applications Based on Endogenous Expression of Tagged Proteins.
Antibody Validation in Bioimaging Applications Based on Endogenous Expression of Tagged Proteins.
复制标题
基于标记蛋白内源表达的生物成像应用中的抗体验证。
DOI：
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发表时间：
2017
影响因子：
4.4
通讯作者：
E. Lundberg
中科院分区：
文献类型：
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作者：
Marie Skogs;Charlotte Stadler;Rutger Schutten;Martin Hjelmare;Christian Gnann;Lars Björk;I. Poser;A. Hyman;M. Uhlén;E. Lundberg
抗体是不可或缺的研究工具，但科学界尚未采用标准化程序来验证其特异性。在这里，我们提出了一种策略，系统地验证抗体的免疫荧光（IF）应用，使用基因标签。我们已经评估了197种抗体的在靶和脱靶结合能力，使用108种内源性表达EGFP标记的靶蛋白的细胞系。此外，我们评估了35种靶蛋白的批次间效应，表明抗体批次之间的靶向和脱靶结合模式差异显著，并且所提出的策略可作为确保新抗体批次生产重现性的可靠程序。总之，我们提出了一个系统的计划，在IF应用中使用内源性表达的标记蛋白的抗体验证。这是迈向可重复方法的重要一步，用于背景和应用特异性抗体验证，并提高基于抗体的实验和研究数据的可靠性。
Antibodies are indispensible research tools, yet the scientific community has not adopted standardized procedures to validate their specificity. Here we present a strategy to systematically validate antibodies for immunofluorescence (IF) applications using gene tagging. We have assessed the on- and off-target binding capabilities of 197 antibodies using 108 cell lines expressing EGFP-tagged target proteins at endogenous levels. Furthermore, we assessed batch-to-batch effects for 35 target proteins, showing that both the on- and off-target binding patterns vary significantly between antibody batches and that the proposed strategy serves as a reliable procedure for ensuring reproducibility upon production of new antibody batches. In summary, we present a systematic scheme for antibody validation in IF applications using endogenous expression of tagged proteins. This is an important step toward a reproducible approach for context- and application-specific antibody validation and improved reliability of antibody-based experiments and research data.
影响因子：
64.5
作者：
Hein, Marco Y.;Hubner, Nina C.;Mann, Matthias
通讯作者：
Mann, Matthias
影响因子：
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作者：
Rizzo, Mark A;Davidson, Michael W;Piston, David W
通讯作者：
Piston, David W