Antibody Validation in Bioimaging Applications Based on Endogenous Expression of Tagged Proteins

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• 作者：Marie Skogs ; Charlotte Stadler ; Rutger Schutten ; Martin Hjelmare ; Christian Gnann ; Lars Björk ; Ina Poser ; Anthony Hyman ; Mathias Uhlén ; Emma Lundberg
• 刊名：Journal of Proteome Research
• 出版年：2017
• 出版时间：January 6, 2017
• 年：2017
• 卷：16
• 期：1
• 页码：147-155
• 全文大小：484K
• ISSN：1535-3907

文摘

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.