Three part assay for kinase or phosphatase activity

Abstract

The present invention relates to a method of determining kinase or phosphatase activity based on a three parts system. The method comprises contacting a binding partner which can bind phosphorylated peptides, a detection molecule and a substrate peptide. Determination of activities is achieved by measuring energy transfer between an energy donor and an energy acceptor that are present on the detection molecule and the substrate molecule.

1 . A method for determining kinase activity of a kinase comprising: 
 a) phosphorylating a substrate molecule that is labelled with an energy donor with a kinase;    b) contacting the phosphorylated substrate molecule obtained in step b) with 
 i) a binding partner that only binds phosphorylated substrate molecule and  
 ii) a detection molecule, wherein the detection molecule is labelled with an energy acceptor, 
 such that the contacting of said substrate molecule with said binding partner and said detection molecule binds said detection molecule to said binding partner; and  
 
   c) determining kinase activity by measuring energy transfer from the energy donor of said substrate molecule to the energy acceptor of said detection molecule bound to said binding partner,    wherein the energy donor has an emission lifetime of less than 10 microseconds.    
   
   
       2 . The method of  claim 1  wherein the energy donor is a Ru-luminophore and the energy acceptor is a flourophore exhibiting a spectral overlap with the Ru-complexes yielding an Ro of at least 10 angstroms.  
   
   
       3 . A method for determining kinase activity of a kinase comprising: 
 a) phosphorylating a substrate molecule that is labelled with an energy acceptor with a kinase;    b) contacting the phosphorylated substrate molecule obtained in step b) with 
 i) a binding partner that only binds phosphorylated substrate molecule and  
 ii) a detection molecule, wherein the detection molecule is labelled with an energy donor, 
 such that the contacting of said substrate molecule with said binding partner and said detection molecule binds said detection molecule to said binding partner; and  
 
   c) determining kinase activity by measuring energy transfer from the energy donor of said detection molecule bound to said binding partner to the energy acceptor of said substrate molecule,    wherein the energy donor has an emission lifetime of less than 10 microseconds.    
   
   
       4 . The method of  claim 3  wherein the energy donor is a Ru-luminophore and the energy acceptor is a flourophore exhibiting a spectral overlap with the Ru-complexes yielding an Ro of at least 10 angstroms.  
   
   
       5 . A method for determining phosphatase activity of a phosphatase comprising 
 a) dephosphorylating a phosphorylated substrate molecule that is labelled with an energy donor with a phosphatase;    b) contacting the dephosphorylated substrate molecule obtained in step a) with 
 i) a binding partner that only binds phosphorylated substrate molecule and  
 ii) a detection molecule, wherein said detection molecule is labled with an energy acceptor,  
 such that the contacting of said substrate molecule with said binding partner and said detection molecule binds said detection molecule to said binding partner; and  
   c) determining phosphatase activity by measuring energy transfer from the energy donor of said substrate molecule to the energy acceptor of said detection molecule bound to said binding partner,    wherein the energy donor has an emission lifetime of less than 10 microseconds.    
   
   
       6 . The method of  claim 5  wherein the energy donor is a Ru-luminophore and the energy acceptor is a flourophore exhibiting a spectral overlap with the Ru-complexes yielding an Ro of at least 10 angstroms.  
   
   
       7 . A method for determining phosphatase activity of a phosphatase comprising 
 a) dephosphorylating a phosphorylated substrate molecule that is labeled with an energy acceptor with a phosphatase;    b) contacting the dephosphorylated substrate molecule obtained in step a) with 
 i) a binding partner that only binds phosphorylated substrate molecule and  
 ii) a detection molecule, wherein said detection molecule is labeled with an energy donor,  
 such that the contacting of said substrate molecule with said binding partner and said detection molecule binds said detection molecule to said binding partner; and  
   c) determining phosphatase activity by measuring energy transfer from the energy donor of said detection molecule bound to said binding partner to the energy acceptor of said substrate molecule,    wherein the energy donor has an emission lifetime of less than 10 microseconds.    
   
   
       8 . The method of  claim 7  wherein the energy donor is a Ru-luminophore and the energy acceptor is a flourophore exhibiting a spectral overlap with the Ru-complexes yielding an Ro of at least 10 angstroms.  
   
   
       9 . A kit for performing the method of  claim 1 ,  3 ,  5  and  7 , comprising, in separate containers, a binding partner, a substrate molecule and at least one detection molecule, wherein the optimal amount of said binding partner and detection molecule can be titrated individually.  
   
   
       10 . A kit for performing the method of claims  1 ,  3 ,  5  and  7 , comprising, in separate containers, binding partner, more than one detection molecule and more than one substrate peptide, wherein the binding partner are metal ions on plastic beads and further wherein said detection molecules and said substrate peptides are provided as sets of FRET pairs.