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

Nat Protoc. 2007;2(5):1145-51. doi: 10.1038/nprot.2007.172.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / isolation & purification*
  • Chromatography, Affinity / methods*
  • Chromosomes, Artificial, Bacterial / genetics
  • Electrophoresis, Polyacrylamide Gel
  • HeLa Cells
  • Humans
  • Mass Spectrometry
  • Mice
  • Multiprotein Complexes / isolation & purification*
  • RNA Interference

Substances

  • Cell Cycle Proteins
  • Multiprotein Complexes
  • SGO1 protein, human