Units Required for Phosphate Removal

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Unit assays for protein phosphatases use the organic substrate p-nitrophenyl phosphate, PNPP (NEB #P0757). The reactivity of phosphatases toward phosphorylated protein substrates can be markedly different from that observed with PNPP. Accordingly, removal of an equivalent amount of phosphate groups from a given protein may require a radically different number of PNPP units to be added to the reaction. The Protein Phosphatases Specificity Chart illustrates this point, indicating the number of units required to completely remove 0.5 nmol phosphate groups from myelin basic protein in a 30 minute reaction under the recommended reaction conditions. Because phosphate removal can differ among proteins, the table lists a range between 5 and 10-fold higher than this determined value to ensure complete dephosphorylation.