Tandem Affinity Purification of Proteins

Arthur Günzl1, Bernd Schimanski2

1 Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut, 2 Institute of Cell Biology, University of Bern, Bern, Switzerland
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.19
DOI:  10.1002/0471140864.ps1919s55
Online Posting Date:  February, 2009
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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

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Tandem Affinity Purification of PTP‐Tagged Proteins from Crude Cell‐Free Extract
  • Basic Protocol 2: Concentrating Tandem Affinity–Purified Proteins and Preparation of an SDS Sample
  • Alternate Protocol 1: Concentrating the Final TAP Eluate in the Absence of SDS
  • Basic Protocol 3: Analysis of Tandem Affinity Purification by Immunoblotting
  • Basic Protocol 4: Analysis of Tandem Affinity Purification by SDS‐PAGE
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Tandem Affinity Purification of PTP‐Tagged Proteins from Crude Cell‐Free Extract

  Materials
  • IgG Sepharose 6 Fast Flow matrix (GE Healthcare)
  • PA‐150 buffer (see recipe)
  • Complete Mini, EDTA‐free protease inhibitor cocktail tablets (Roche)
  • Cell extract for analysis (see ; buffer used should resemble PA‐150 buffer, see recipe, in pH and salt conditions)
  • 10% (v/v) Tween 20 (Sigma‐Aldrich)
  • Liquid nitrogen
  • TEV protease buffer (see recipe)
  • PC‐150 buffer (see recipe)
  • AcTEV protease (Invitrogen)
  • Anti‐Protein C (anti‐ProtC) affinity matrix (Roche)
  • 1 M CaCl 2 stock solution
  • EGTA elution buffer (see recipe)
  • Protein C peptide (EDQVDPRLIDGK; synthesized in‐house)
  • Peptide elution buffer (see recipe)
  • 0.8 × 4–cm Poly‐Prep chromatography columns (Bio‐Rad)
  • Adjustable stand to hold columns
  • End‐over‐end rotator
  • Scalpel
NOTE: Unless otherwise specified, all steps should be carried out at 4°C. All buffers should be cooled on ice prior to use.

Basic Protocol 2: Concentrating Tandem Affinity–Purified Proteins and Preparation of an SDS Sample

  Materials
  • Solution of TAP‐purified protein (EGTA eluate; protocol 1, step 14a)
  • StrataClean resin (Stratagene)
  • 1× SDS gel loading buffer (see recipe)
  • Vacuum concentrator (e.g., SpeedVac evaporator)
  • End‐over‐end rotator
  • Minifuge (Harvard Apparatus; or other microcentrifuge)
  • 100°C water bath

Alternate Protocol 1: Concentrating the Final TAP Eluate in the Absence of SDS

  • 10% (v/v) Tween 20 (Sigma‐Aldrich)
  • Dialysis buffer (see recipe)
  • 1‐liter glass beaker
  • Vacuum concentrator (e.g., SpeedVac evaporator)
  • Standard floating device for microcentrifuge tubes
  • Mini dialysis units (Slide‐A‐Lyzer, 3500 MWCO, Pierce)

Basic Protocol 3: Analysis of Tandem Affinity Purification by Immunoblotting

  Materials
  • TAP fractions ( protocol 1)
  • 4× SDS gel loading buffer (see recipe)
  • Monoclonal anti‐ProtC antibody HPC4 (Roche)
  • Anti‐mouse IgG antibody, peroxidase conjugated (Vector Laboratories)
  • BM chemiluminescent blotting substrate (Roche)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1), immunoblotting (units 10.7), and immunoblot detection (unit 10.10)

Basic Protocol 4: Analysis of Tandem Affinity Purification by SDS‐PAGE

  Materials
  • TAP fractions ( protocol 1)
  • 1× SDS gel loading buffer (see recipe)
  • GelCode blue stain reagent (Pierce)
  • Additional reagents and equipment for collecting proteins with StrataClean resin beads ( protocol 2, steps 2 to 5) and SDS‐PAGE (unit 10.1)
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Figures

Videos

Literature Cited

Literature Cited
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