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 language | English (US) |
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Pages (from-to) | Unit 20.2 |
Journal | Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.] |
Volume | Chapter 20 |
DOIs | |
State | Published - May 2001 |
Externally published | Yes |
ASJC Scopus subject areas
- Molecular Biology