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)|
|Pages (from-to)||Unit 20.2|
|Journal||Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]|
|State||Published - May 2001|
ASJC Scopus subject areas
- Molecular Biology