Kinases can be obtained as both pure (recombinant) enzymes, or as relatively crude lysates. The source of the enzyme may affect the choice of substrate. For example, when using a highly purified recombinant kinase, a substrate protein or peptide can be specific or generic, because only one kinase activity is present. In a crude lysate, there may be more than one kinase activity present. In this instance, a generic substrate may be phosphorylated by a number of different kinases, whereas a specific substrate will give information only about the kinase of interest.
Enzymes must be available in an activated form, whether they are recombinant or in lysates. Does the reaction buffer include known co-factors?
SPA kinase assays have been developed with a wide range of kinases, including serine/threonine, tyrosine, and histidine. Some kinases are known to autophosphorylate. To avoid this affecting assay performance, pre-incubation in the absence of labelled ATP can eliminate increased background due to radiolabelling of the kinase.
When using relatively crude kinase preparations it is highly likely that a competing phosphatase will also be present. An inhibitor should be included. Proteases may also be present and thus protease inhibitors should be included to prevent kinase or substrate degradation.