Systematic validation of antibody binding and protein subcellular localization using siRNA and confocal microscopy
Systematic validation of antibody binding and protein subcellular localization using siRNA and confocal microscopy
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DOI：
10.1016/j.jprot.2012.01.030
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发表时间：
2012-04-03
影响因子：
3.3
通讯作者：
Lundberg, Emma
中科院分区：
文献类型：
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作者：
Stadler, Charlotte;Hjelmare, Martin;Lundberg, Emma
我们已经开发了一个平台，用于验证抗体结合和蛋白质亚细胞定位数据，从免疫荧光使用siRNA技术结合自动共聚焦显微镜和图像分析。通过将siRNA技术与自动化样品制备、自动化成像和定量图像分析相结合，已经建立了高通量测定，以系统地确认准确的蛋白质结合和定位。在这里，我们描述了65种人类蛋白质的亚细胞定位的分析和验证，这些蛋白质被75种抗体靶向，并被130种siRNA沉默。大部分（80%）亚细胞位置，包括几种先前未表征的蛋白质的位置，可以通过siRNA沉默后抗体信号的显著下调来证实。使用自动图像分析建立定量分析，以便于研究在多个隔室中发现的靶点。使用该平台获得的结果表明，siRNA沉默与细胞不同区室中抗体信号的定量图像分析相结合是一种有吸引力的方法，用于确保准确的蛋白质定位以及使用免疫荧光的抗体结合。由于人类蛋白质组的大部分尚未被探索，我们认为这种方法在亚细胞水平上绘制人类蛋白质组的持续工作中具有重要意义。(C)2012爱思唯尔有限公司版权所有。
We have developed a platform for validation of antibody binding and protein subcellular localization data obtained from immunofluorescence using siRNA technology combined with automated confocal microscopy and image analysis. By combining the siRNA technology with automated sample preparation, automated imaging and quantitative image analysis, a high-throughput assay has been set-up to enable confirmation of accurate protein binding and localization in a systematic manner. Here, we describe the analysis and validation of the subcellular location of 65 human proteins, targeted by 75 antibodies and silenced by 130 siRNAs. A large fraction of (80%) the subcellular locations, including locations of several previously uncharacterized proteins, could be confirmed by the significant down-regulation of the antibody signal after the siRNA silencing. A quantitative analysis was set-up using automated image analysis to facilitate studies of targets found in more than one compartment. The results obtained using the platform demonstrate that siRNA silencing in combination with quantitative image analysis of antibody signals in different compartments of the cells is an attractive approach for ensuring accurate protein localization as well as antibody binding using immunofluorescence. With a large fraction of the human proteome still unexplored, we suggest this approach to be of great importance under the continued work of mapping the human proteome on a subcellular level. (C) 2012 Elsevier B.V. All rights reserved.