Analysis of GAPDH and Protein Interaction

Abstract

The non-glycolytic or non-enzymatic functions of GAPDH also involve its interaction with other proteins. GAPDH has been reported to interact with cytoplasmic as well as nuclear proteins (transcription factors). This chapter will outline methods that can be used to decipher the interaction of GAPDH with other proteins in a cell-free system as well as in vivo. If both GAPDH and the protein of interest are available in sufficient amounts, either in native or recombinant forms, the cell-free system method will be relevant for verification of the GAPDH and protein interaction. The assay relies on the principle of the electrophoretic mobility shift assay (EMSA). This method is quick and simple, yet reliable. If the in vivo interaction of GAPDH with other proteins is to be investigated, then immunoprecipitation of GAPDH is appropriate. In principle, as the GAPDH is immunoprecipitated (pulled-down) any other protein that is bound with GAPDH will also be pulled-down (known as co-immunoprecipitation). Thus, this approach relies on the co-immunoprecipitation method to identify interacting partners. The advantage of the co-immunoprecipitation technique is that it will demonstrate the real-time binding (based on cellular dynamics or events) of GAPDH with a target protein in the context of the cellular status (e.g. proliferation, programmed cell death, disease conditions, etc.).