title: Affinity purification of proteins binding to GST fusion proteins.
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.
authors.affiliation: University of Texas Southwestern Medical Center, Dallas, Texas, USA.
authors.forename: J C
authors.initials: JC
authors.lastname: Swaffield
authors.forename: S A
authors.initials: SA
authors.lastname: Johnston
mesh_terms: 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
chemical_list: D007202:Indicators and Reagents; D011993:Recombinant Fusion Proteins; D012996:Solutions; D005982:Glutathione Transferase