Staining technique for phosphatases in polyacrylamide gels

The described staining technique affords a means to detect and evaluate directly on the electrophoresis polyacrylamide gel the activity of phosphatases present in complex extracts or in purified solutions, in particular phosphatases requiring alkaline pH for optimum activity, e.g. fructose bisphosphatase, sedoheptulose bisphosphatase, and alkaline phosphatase, and 5'-nucleotidase. The method is based on a modified malachite green procedure determining orthophosphate released by the specific enzyme reactions carried out in the gel and leads to the formation of sharp blue-green-colored bands on a pale blue background. Staining intensity is proportional to the amount of Pi formed. The lower limit of phosphate detection was in the order of 0.2 nmol. This method provides a routine tool for comparative studies of biochemical properties and isozymic composition of phosphatases from diverse sources.