Protein Tyrosine Kinase (PTK) is an enzyme critical to the signal transduction pathways that control cell proliferation, differentiation and carcinogenesis. PTKs are classified into two familiess: membrane receptor PTKs such as insulin and EGF receptor and non-receptor linked PTKs such as Src and FAK. Membrane receptor PTKs contain three domains: the extracellular, membrane intrinsic and intracellular domains. The extracellular and membrane intrinsic domains bind to ligands such as hormones and growth factors, while the intracellular domain contains the PTK active site. Receptor PTKs transduce signals directly into the cell through ligand mediated tyrosine phosphorylation. Non-receptor linked PTKs lack the membrane intrinsic domain and are divided into three types based upon their cellular location (i.e., membrane, cytoplasmic or nuclear). Non-receptor linked PTKs participate in various functions, including signal transduction and cell-cell contact responses.
The Universal Tyrosine Kinase Assay Kit is an in vitro enzyme immunoassay kit intended for the quantitative determination of tyrosine kinase activity in either adherent or suspension cell culture extracts. The kit can also be used to measure PTK activity levels and their regulation as well as for the in vitro screening of PTK inhibitors. The kit includes a 96-well microtiter plate coated with PTK substrate, a phosphotyrosine (pY20)-horseradish peroxidase (HRP) antibody, and an HRP substrate (TMBZ). Serial dilutions of prepared cell extract may be added alongside an ATP solution to the PTK substrate-coated microtiter plate; this enables phosphorylation of tyrosine. The sample solution is then removed and the plate thoroughly washed and blocked. Following a blocking step, pY20-HRP is added, incubated, and then replaced by the TMBZ solution. Ensuing color development enables sample PTK activity to be determined by comparing sample absorbance at 450 nm with the absorbance obtained for the standard (via a plotted standard curve).