Optimized Affinity Capture of Yeast Protein Complexes.
Optimized Affinity Capture of Yeast Protein Complexes.
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DOI：
10.1101/pdb.prot087932
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发表时间：
2016-07
影响因子：
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通讯作者：
J. LaCava;Javier Fernandez-Martinez;Zhanna Hakhverdyan;M. Rout
中科院分区：
文献类型：
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作者：
J. LaCava;Javier Fernandez-Martinez;Zhanna Hakhverdyan;M. Rout
在这里，我们描述了一个亲和分离协议。它使用冷冻研磨的酵母细胞粉生产细胞提取物和抗体结合的顺磁珠进行亲和捕获。提供了确定最佳浸提溶剂组合物的指南。捕获的蛋白质在变性溶剂（十二烷基硫酸钠聚丙烯酰胺凝胶电泳样品缓冲液）中洗脱，用于基于凝胶的蛋白质组学分析。虽然可以修改程序以使用其他来源的细胞提取物和其他形式的亲和介质，但迄今为止，我们一直使用所提出的方法获得最佳结果。
Here, we describe an affinity isolation protocol. It uses cryomilled yeast cell powder for producing cell extracts and antibody-conjugated paramagnetic beads for affinity capture. Guidelines for determining the optimal extraction solvent composition are provided. Captured proteins are eluted in a denaturing solvent (sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer) for gel-based proteomic analyses. Although the procedures can be modified to use other sources of cell extract and other forms of affinity media, to date we have consistently obtained the best results with the method presented.