Method for quantifying phosphokinase activity on proteins

Abstract

The invention involves a method for measuring phosphorylation of proteins at specific sites and, as such, is an indicator of the protein kinase activity of enzymes capable of phosphorylating those sites. The method involves the in vitro or in vivo phosphorylation of a target protein at a specific serine, threonine or tyrosine residue, subjecting that protein (non-phosphorylated) to reaction mixture containing all reagents, including phosphokinase which allow the creation of a phosphorylated form of protein. The phosphorylated protein is measured by contacting it with an antibody specific for the phosphorylation site(s). The invention includes antibodies useful in practicing the methods of the invention. The invention particularly relates to all proteins modified by phosphorylation and dephosphorylation as illustrated by Tau, Rb and EGFR proteins and antibodies specific for the site of phosphorylation of the Tau, Rb or EGFR proteins.

1 . A method for measuring phosphokinase activity on Tau protein comprising:
 (a) subjecting a protein to a phosphokinase to phosphorylate a phosphorylation site on the protein;   (b) providing an antibody specific to a phosphorylated phosphorylation site on the protein;   (c) contacting the protein from (a) with the antibody of (b); and   (d) detecting the antibody bound to the phosphorylation site; wherein the phosphorylation site of the protein is a serine.   
     
     
         2 . (canceled) 
     
     
         3 . A method according to  claim 2  wherein the phosphokinase is GSK-3β, PKA, PKC, CDK-5, MARK, JNK, p38MAPK, or casein kinase II. 
     
     
         4 - 7 . (canceled) 
     
     
         8 . An antibody prepared from a polypeptide immunogen having a phosphorylated serine. 
     
     
         9 - 10 . (canceled) 
     
     
         11 . An antibody specific for sequence ID #1. 
     
     
         12 . An antibody specific for sequence ID #2. 
     
     
         13 - 20 . (canceled) 
     
     
         21 . The method of  claim 1  wherein the serine group is S 199  of the protein 
     
     
         22 . The method of  claim 1  wherein the serine group is S 214  of the protein. 
     
     
         23 . The method of  claim 1  wherein the serine group is S 202 , S 396 , or S 404  of the protein.