UNIT 20.2 Affinity Purification of Proteins Binding to GST Fusion Proteins
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Swaffield, J. C. and Johnston, S. A. 2001. Affinity Purification of Proteins Binding to GST Fusion Proteins. Current Protocols in Molecular Biology. 33:20.2:20.2.1–20.2.10.
- Published Online: 1 MAY 2001
- Published Print: JAN 1996
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 Basic Protocol 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 Support Protocol describes preparation of an E. coli extract that is added to the reaction mixture with purified test protein to reduce nonspecific binding.