Affinity Purification of Proteins Binding to GST Fusion Proteins

Jonathan C. Swaffield1, Stephen Albert Johnston1

1 University of Texas Southwestern Medical Center, Dallas, Texas
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 20.2
DOI:  10.1002/0471142727.mb2002s33
Online Posting Date:  May, 2001
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Abstract

This unit describes the use of proteins fused to glutathione‐S‐transferase (GST fusion proteins) to affinity purify other proteins, a technique also known as GST pulldown purification. The describes a strategy in which a GST fusion protein is bound to agarose affinity beads and the complex is then used to assay the binding of a specific test protein that has been labeled with [35S]methionine by in vitro translation. However, this method can be adapted for use with other types of fusion proteins; for example, His6, biotin tags, or maltose‐binding protein fusions (MBP), and these may offer particular advantages. A describes preparation of an E. coli extract that is added to the reaction mixture with purified test protein to reduce nonspecific binding.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: GST Fusion Protein–Affinity Purification
  • Support Protocol 1: Preparation of E. Coli Extract
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: GST Fusion Protein–Affinity Purification

  Materials
  • E. coli extract in bead binding buffer (see protocol 2)
  • Test protein labeled with [35S]methionine (unit 10.17)
  • 100 to 250 µg/ml GST and GST fusion protein bound to agarose beads (unit 16.7), freshly prepared
  • SDS sample buffer (unit 10.2)
  • Gel fixative: 10% (v/v) glacial acetic acid/45% (v/v) methanol
  • Centrifuge and Beckman TLA‐100 rotor or equivalent
  • Microcentrifuge, 4°C
  • X‐ray film or Storage Phosphor screen (Molecular Dynamics)
  • Scanning densitometer or phosphorimager
  • Additional reagents and equipment for SDS‐PAGE (unit 10.2), Coomassie blue staining (unit 10.6; optional), and autoradiography ( appendix 3A)

Support Protocol 1: Preparation of E. Coli Extract

  Materials
  • E. coli overnight culture grown in LB medium (unit 1.1)
  • recipeBead binding buffer (see recipe) with and without Triton X‐100 and glycerol, 4°C
  • Tween 20
  • Glycerol
  • Sonicator with microprobe
  • Centrifuge and Sorvall SS‐34 rotor or equivalent
  • Additional reagents and equipment for growing E. coli (unit 1.2) and quantitating proteins in solution (unit 10.1)
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Figures

Videos

Literature Cited

Literature Cited
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Key Reference
   Melcher, K. and Johnston, S.A. 1995. Gal4 interacts with TATA‐binding protein and co‐activators. Mol. Cell Biol. 15:2839‐2848.
  Describes binding from extracts, binding of in vitro‐translated and labeled protein, binding‐deficient mutants, correlation of in vitro and in vivo results, and determination of binding constant.
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