Affinity purification of proteins binding to GST fusion proteins.
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.
Swaffield
J C
JC
University of Texas Southwestern Medical Center, Dallas, Texas, USA.
Johnston
S A
SA
D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics*; D007202:Indicators and Reagents; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D012996:Solutions
D007202:Indicators and Reagents; D011993:Recombinant Fusion Proteins; D012996:Solutions; D005982:Glutathione Transferase