A proteomics approach for the identification of DNA binding activities observed in the electrophoretic mobility shift assay
A proteomics approach for the identification of DNA binding activities observed in the electrophoretic mobility shift assay
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DOI：
10.1074/mcp.t200003-mcp200
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发表时间：
2002-06-01
影响因子：
7
通讯作者：
Abraham, LJ
中科院分区：
文献类型：
--
作者：
Woo, AJ;Dods, JS;Abraham, LJ
转录因子位于基因调控的中心，它们的识别对于理解转录和基因表达至关重要。传统上，转录因子的分离和鉴定是一项漫长而艰巨的任务。我们在此提出了一种利用二维电泳与质谱联用的能力来鉴定电泳迁移率变动分析 (EMSA) 中的 DNA 结合蛋白的新方法。通过将 SDS-PAGE 和等电聚焦与 EMSA 结合，可以估计 EMSA 中看到的蛋白质复合物的分子质量和 pi。然后在二维阵列上以预定的 pl 和分子质量坐标鉴定候选蛋白质，并通过质谱法鉴定。我们在此展示了功能相关转录因子的成功分离，并通过 EMSA 超位移分析验证了其身份。
Transcription factors lie at the center of gene regulation, and their identification is crucial to the understanding of transcription and gene expression. Traditionally, the isolation and identification of transcription factors has been a long and laborious task. We present here a novel method for the identification of DNA-binding proteins seen in electrophoretic mobility shift assay (EMSA) using the power of two-dimensional electrophoresis coupled with mass spectrometry. By coupling SDS-PAGE and isoelectric focusing to EMSA, the molecular mass and pi of a protein complex seen in EMSA were estimated. Candidate proteins were then identified on a two-dimensional array at the predetermined pl and molecular mass coordinates and identified by mass spectrometry. We show here the successful isolation of a functionally relevant transcription factor and validate the identity through EMSA super-shift analysis.