Native gel activity stain and preparative electrophoretic method for the detection and purification of pyridine...

An activity stain for the detection of pyridine nucleotide-linked dehydrogenases in polyacrylamide gels is described. Following incubation of the gel with substrate and cofactor, bands are visualized under ultraviolet light, where reduced cofactors fluoresce and oxidized cofactors appear black. The methods described are useful for any NAD- or NADP-linked dehydrogenase; the enzymes can be assayed in either the oxidative or the reductive direction. Also described is a preparative polyacrylamide gel system using the activity stain, which can be used as a general purification method for dehydrogenases. The preparative gels are crosslinked with bisacrylylcystamine. These crosslinks can be broken by the addition of thiols after the bands of interest have been located and excised. The protein of interest is then separated from the solubilized acrylamide by adsorption to a suitable resin.