In vitro fluorescence polarization assay

An in vitro assay method for identifying a test substance which competitively binds to either a receptor tyrosine-phosphorylated peptide and/or its ligand, said method comprising:

• (a) providing (i) a receptor for a tyrosine-phosphorylated peptide, and (ii) a ligand for said receptor, said ligand bearing a covalently linked fluorescent moiety,
• (b) irradiating a mixture containing (i), (ii) and said test substance with polarized light of a suitable wavelength permitting excitation of the fluorophore as indicated by emission of polarized light,
• (c) measuring the degree of polarization of the emission, and
• (d) determining the effect of the presence or concentration of the test substance in decreasing the observed emission polarization of a mixture of (i) and (ii) alone, wherein competitive binding of said test substance correlates with decreased depolarization values.

An in vitro assay method for identifying a test substance which inhibits the mutual association of two protein molecules, said method comprising:

• (a) providing a first protein molecule and a second protein molecule capable of mutual association, said second protein molecule bearing a covalently linked fluorophore,
• (b) preparing a mixture containing said first and second protein molecules and at least one test substance,
• (c) irradiating said mixture with polarized light of a suitable wavelength permitting excitation of the fluorophore as indicated by emission of polarized light,
• (d) measuring the degree of polarization of the emission, and
• (e) determining the effect of the presence or concentration of the test substance in decreasing the observed emission polarization of a mixture of said first and second protein molecules in the absence of said test substance.