Isolation of Novel Substrates Using a Tyrosine Kinase Overlay/In Situ Assay

Identifying substrates of receptor and non-receptor protein tyrosine kinases (PTK), and how phosphorylation of these substrates affects signaling and cytoskeletal pathways, has been a key step in understanding the role of PTK in differentiation, mitogenesis and oncogenesis. However, it has been difficult to distinguish substrates phosphorylated directly by PTK vs those phosphorylated by PTK-activated kinases. The following describes an in situ /overlay technique in which purified PTK (in our case, FAK) can be used to identify potential substrates from filter lifts of protein produced from a cDNA expression library.