Analysis of GAPDH – Native Protein

Abstract

GAPDH protein analysis under native, non-denatured condition combined with immunoblotting is a valid tool to determine the overall or total molecular mass of GAPDH protein, without involving cumbersome purification or isolation processes. It is one of the fast and reliable approaches as it involves the identification or recognition of GAPDH using specific antibody. Physiologically, as the total molecular mass includes the mass contributed by individual subunits the findings could reveal any cell-specific or pathway-specific posttranslational modification of subunits by phosphorylation, glycation and so on. Besides, it may also show any aggregation or modification due to covalent binding with other intracellular components. More importantly, any biochemical change (e.g. phosphorylation) in GAPDH would impact its pI (isoelectric point) as well which in turn may impact its cellular function. Thus, native-gel analysis of GAPDH followed by immunoblotting is an effective tool to understand any change in GAPDH as an intact, native protein. In this chapter we will describe relevant procedures pertinent to the identification of GAPDH by immunoblotting.