Tandem affinity purification of functional TAP-tagged proteins from human cells

被引：44
作者：
Gregan, Juraj [1 ,2 ]
Riedel, Christian G. [2 ]
Petronczki, Mark [2 ]
Cipak, Lubos [1 ]
Rumpf, Cornelia [1 ]
Poser, Ina [3 ]
Buchholz, Frank [3 ]
Mechtler, Karl [2 ]
Nasmyth, Kim [2 ,4 ]
机构：
[1] Univ Vienna, Dept Chrosome Biol, Max F Perutz Labs, A-1030 Vienna, Austria
[2] Res Inst Mol Pathol, A-1030 Vienna, Austria
[3] Max Planck Inst Mol Cell Biol & Genet, D-01307 Dresden, Germany
[4] Univ Oxford, Dept Biochem, Oxford OX1 3QU, England
来源：
NATURE PROTOCOLS | 2007年 / 2卷 / 05期
基金：
奥地利科学基金会;
关键词：
D O I：
10.1038/nprot.2007.172
中图分类号：
Q5 [生物化学];
学科分类号：
071010 ; 081704 ;
摘要：
Tandem affinity purification ( TAP) is a generic two-step affinity purification protocol for isolation of TAP-tagged proteins together with associated proteins. We used bacterial artificial chromosome to heterologously express TAP-tagged murine Sgo1 protein in human HeLa cells. This allowed us to test the functionality of the Sgo1-TAP protein by RNA interference-mediated depletion of the endogenous human Sgo1. Here, we present an optimized protocol for purification of TAP-tagged Sgo1 protein as well as KIAA1387 from HeLa cells with detailed instructions. The purification protocol can be completed in 1 day and it should be applicable to other proteins.
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收藏
页码：1145 / 1151
页数：7
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