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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.

Original publication

DOI

10.1038/nprot.2007.172

Type

Journal article

Journal

Nat Protoc

Publication Date

2007

Volume

2

Pages

1145 - 1151

Keywords

Animals, Cell Cycle Proteins, Chromatography, Affinity, Chromosomes, Artificial, Bacterial, Electrophoresis, Polyacrylamide Gel, HeLa Cells, Humans, Mass Spectrometry, Mice, Multiprotein Complexes, RNA Interference