Unit

UNIT 19.19 Tandem Affinity Purification of Proteins

  1. Arthur Günzl1,
  2. Bernd Schimanski2

Published Online: 1 FEB 2009

DOI: 10.1002/0471140864.ps1919s55

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Günzl, A. and Schimanski, B. 2009. Tandem Affinity Purification of Proteins. Current Protocols in Protein Science. 55:19.19:19.19.1–19.19.16.

Author Information

  1. 1

    Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut

  2. 2

    Institute of Cell Biology, University of Bern, Bern, Switzerland

Publication History

  1. Published Online: 1 FEB 2009
  2. Published Print: FEB 2009

Abstract

Tandem affinity purification (TAP) is a very efficient method to isolate proteins, protein complexes, or ribonucleoprotein particles from crude extracts. The method depends on the expression of one protein component fused N- or C-terminally to a TAP tag in the organism of interest. The TAP tag is a composite tag consisting of two different epitope domains and a protease cleavage site, and it facilitates the purification of the tagged protein in two consecutive, high-affinity chromatography steps. Combined, the two steps are typically so efficient that a protein complex can be purified virtually to homogeneity without the need for protein overexpression. If the tag does not interfere with protein function, TAP is likely to yield an intact protein complex because all steps of the procedure are carried out under nondenaturing conditions. In this unit, a TAP procedure is detailed which employs a novel epitope combination termed PTP. Curr. Protoc. Protein Sci. 55:19.19.1-19.19.16. © 2009 by John Wiley & Sons, Inc.

Keywords:

  • tandem affinity purification;
  • TAP;
  • PTP tag;
  • protein A domain;
  • protein C epitope;
  • tobacco etch virus (TEV) protease;
  • IgG chromatography;
  • anti-protein C immunoaffinity chromatography