Protocol

Far Western: Labeling GST Fusion Proteins

This protocol was adapted from "Identification of Protein-Protein Interactions with Glutathione-S-Transferase Fusion Proteins," Chapter 6, in Protein-Protein Interactions, 2nd edition (eds. Golemis and Adams). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION

A far-Western blot (also known as an overlay assay) is used to detect the interaction of a recombinant GST fusion protein (produced and purified from bacteria) with a target protein on a membrane. Three methods are generally used to detect an interaction: radioactive labeling of the fusion protein, biotinylation of the fusion protein, and detection by anti-GST antibodies. This protocol describes the radioactive labeling of GST fusion proteins using a phosphorylation site that has been integrated into the fusion protein. This is rapid, easy, and because the phosphorylation site is in the fusion portion of the protein, labeling the fusion protein generally has little impact on subsequent activity. The fusion protein consists of a GST moiety, a protease cleavage site, and the phosphorylation target site for a known kinase, which are translated in-frame with the protein of interest. The purified protein is bound to glutathione beads and is radioactively labeled with ³²P using a commercially available kinase. Unincorporated nucleotides are removed from the solution by washing, and the radioactively labeled protein is cleaved with protease (e.g., factor X or thrombin) or eluted with glutathione to remove the GST moiety, which eliminates the possibility of detecting proteins bound to GST during membrane probing.