Substrate phosphorylation by protein kinases on ProtoArray®. (a) Examples of substrate phosphorylations on protein microarrays in the presence of p38alpha and ROKII kinases. (b) Solution validation of commercial kinase substrate pairs. Square box indicates substrate phosphorylation by corresponding kinase. (c) Comparison of different kinase buffers on substrate phosphorylation., Phosphorylation of inactive p38alpha by MKK6 was observed only in Buffer system I (BSA). Tau protein was phosphorylated by PKA to a greater degree in Buffer System II (BSA-free). Net signal was determined by calculating a ratio for the [Kinase-ATP only]BufferI/[Kinase-ATP only]BufferII (d) Venn diagram of substrates phosphorylated by kinases in different buffer systems.