Kinase receptor activation assay

摘要

An assay for measuring activation (i.e., autophosphorylation) of a tyrosine kinase receptor of interest is disclosed. (a) A first solid phase is coated with a substantially homogeneous population of cells so that the cells adhere to the first solid phase. The cells have either an endogenous tyrosine kinase receptor or have been transformed with DNA encoding a receptor or "receptor construct" and the DNA has been expressed so that the receptor or receptor construct is presented in the cell membranes of the cells. (b) A ligand is then added to the solid phase having the adhering cells, such that the tyrosine kinase receptor is exposed to the ligand. (c) Following exposure to the ligand, the adherent cells are solubilized, thereby releasing cell lysate. (d) A second solid phase is coated with a capture agent which binds specifically to the tyrosine kinase receptor, or, in the case of a receptor construct, to the flag polypeptide. (e) The cell lysate obtained in step (c) is added to the wells containing the adhering capture agent so as to capture the receptor or receptor construct to the wells. (f) A washing step is then carried out, so as to remove unbound cell lysate, leaving the captured receptor or receptor construct. (g) The captured receptor or receptor construct is exposed to a labelled antiphosphotyrosine antibody which identifies phosphorylated residues in the tyrosine kinase receptor. (h) Binding of the anti-phosphotyrosine antibody to the captured receptor or receptor construct is measured.