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.
引用
收藏
页码：1145 / 1151
页数：7
相关论文
共 23 条
  • [11] A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins
    Ishizuka, A
    Siomi, MC
    Siomi, H
    [J]. GENES & DEVELOPMENT, 2002, 16 (19) : 2497 - 2508
  • [12] Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search
    Keller, A
    Nesvizhskii, AI
    Kolker, E
    Aebersold, R
    [J]. ANALYTICAL CHEMISTRY, 2002, 74 (20) : 5383 - 5392
  • [13] Human Bub1 defines the persistent cohesion site along the mitotic chromosome by affecting shugoshin localization
    Kitajima, TS
    Hauf, S
    Ohsugi, M
    Yamamoto, T
    Watanabe, Y
    [J]. CURRENT BIOLOGY, 2005, 15 (04) : 353 - 359
  • [14] RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells
    Kittler, R
    Pelletier, L
    Ma, CL
    Poser, I
    Fischer, S
    Hyman, AA
    Buchholz, F
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2005, 102 (07) : 2396 - 2401
  • [15] An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division
    Kittler, R
    Putz, G
    Pelletier, L
    Poser, I
    Heninger, AK
    Drechsel, D
    Fischer, S
    Konstantinova, I
    Habermann, B
    Grabner, H
    Yaspo, ML
    Himmelbauer, H
    Korn, B
    Neugebauer, K
    Pisabarro, MT
    Buchholz, F
    [J]. NATURE, 2004, 432 (7020) : 1036 - 1040
  • [16] Global landscape of protein complexes in the yeast Saccharomyces cerevisiae
    Krogan, NJ
    Cagney, G
    Yu, HY
    Zhong, GQ
    Guo, XH
    Ignatchenko, A
    Li, J
    Pu, SY
    Datta, N
    Tikuisis, AP
    Punna, T
    Peregrín-Alvarez, JM
    Shales, M
    Zhang, X
    Davey, M
    Robinson, MD
    Paccanaro, A
    Bray, JE
    Sheung, A
    Beattie, B
    Richards, DP
    Canadien, V
    Lalev, A
    Mena, F
    Wong, P
    Starostine, A
    Canete, MM
    Vlasblom, J
    Wu, S
    Orsi, C
    Collins, SR
    Chandran, S
    Haw, R
    Rilstone, JJ
    Gandi, K
    Thompson, NJ
    Musso, G
    St Onge, P
    Ghanny, S
    Lam, MHY
    Butland, G
    Altaf-Ui, AM
    Kanaya, S
    Shilatifard, A
    O'Shea, E
    Weissman, JS
    Ingles, CJ
    Hughes, TR
    Parkinson, J
    Gerstein, M
    [J]. NATURE, 2006, 440 (7084) : 637 - 643
  • [17] Shugoshin prevents dissociation of cohesin from centromeres during mitosis in vertebrate cells
    McGuinness, BE
    Hirota, T
    Kudo, NR
    Peters, JM
    Nasmyth, K
    [J]. PLOS BIOLOGY, 2005, 3 (03) : 433 - 449
  • [18] A statistical model for identifying proteins by tandem mass spectrometry
    Nesvizhskii, AI
    Keller, A
    Kolker, E
    Aebersold, R
    [J]. ANALYTICAL CHEMISTRY, 2003, 75 (17) : 4646 - 4658
  • [19] The tandem affinity purification (TAP) method:: A general procedure of protein complex purification
    Puig, O
    Caspary, F
    Rigaut, G
    Rutz, B
    Bouveret, E
    Bragado-Nilsson, E
    Wilm, M
    Séraphin, B
    [J]. METHODS, 2001, 24 (03) : 218 - 229
  • [20] Protein phosphatase 2A protects centromeric sister chromatid cohesion during meiosis I
    Riedel, CG
    Katis, VL
    Katou, Y
    Mori, S
    Itoh, T
    Helmhart, W
    Gálova, M
    Petronczki, M
    Gregan, J
    Cetin, B
    Mudrak, I
    Ogris, E
    Mechtler, K
    Pelletier, L
    Buchholz, F
    Shirahige, K
    Nasmyth, K
    [J]. NATURE, 2006, 441 (7089) : 53 - 61
123