Patent ID: 6203994
Filing Date: 2001-03-20
Classification: C12Q,G01N,Y10S

Abstract:
A method of determining the phosphorylating activity of an enzyme comprising the steps of:(a) combining said enzyme with(i) a reporter molecule comprising a fluorescent label and a phosphorylated amino acid, wherein said phosphorylated amino acid is selected from the group consisting of the phosphorylated form of serine, threonine and tyrosine;(ii) a substrate molecule comprising the same amino acid in its un-phosphorylated state that corresponds to said phosphorylated amino acid in said reporter, wherein said substrate molecule is capable of being phosphorylated at said amino acid by said enzyme to yield a product;(iii) an antibody which selectively binds to said reporter molecule and a phosphorylated substrate molecule both comprising said phosphorylated amino acid(iv) a phosphate source;(b) measuring fluorescence polarization, fluorescence quenching, or fluorescence correlation spectroscopy of said reporter following the combination of step (a); and(c) using fluorescence polarization, fluorescence quenching, or fluorescence correlation spectroscopy measurement of step (b) to determine the activity of said enzyme.