Protocol

Detection of Protein-Protein Interactions Using Far Western with GST Fusion Proteins

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

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INTRODUCTION

Far western analysis was developed to screen cDNA expression libraries for clones that can interact with a ³²P-labeled target protein fused to GST. The target-GST fusion protein is synthesized in bacteria, purified by affinity chromatography on glutathione agarose beads, and labeled in an in vitro reaction catalyzed by a commercially available protein kinase. The fusion protein is then digested with a protease to remove the GST moiety, and the labeled target is used to probe an expression library and/or membranes containing putative interacting proteins that have been separated by SDS-PAGE. This protocol was provided by Margret B. Einarson (Fox Chase . . . [Full Text of this Article]

MATERIALS

METHOD