Protocol abstract
Nature Protocols 2, - 1145 - 1151 (2007)
Published online: 3 May 2007 | doi:10.1038/nprot.2007.172
Subject Categories: Isolation, purification and separation | Biochemistry and protein analysis
Tandem affinity purification of functional TAP-tagged proteins from human cells
Juraj Gregan1,2, Christian G Riedel2, Mark Petronczki2, Lubos Cipak1, Cornelia Rumpf1, Ina Poser3, Frank Buchholz3, Karl Mechtler2 & Kim Nasmyth2,4
Abstract
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.
- Department of Chromosome Biology, Max F. Perutz Laboratories, University of Vienna, Dr Bohr-Gasse 1, 1030 Vienna, Austria.
- Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, 1030 Vienna, Austria.
- Max-Planck-Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany.
- Department of Biochemistry, University of Oxford, South Parks Road, OX1 3QU Oxford, UK.
Correspondence to: Juraj Gregan1,2 e-mail: juraj.gregan@univie.ac.at
