Affinity purification of proteins binding to GST fusion proteins.

J. C. Swaffield, S. A. Johnston

Research output: Contribution to journalArticle

2 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)Unit 20.2
JournalCurrent protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]
VolumeChapter 20
DOIs
StatePublished - May 2001
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Biology

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