Compositions and methods for detection and isolation of phosphorylated molecules

A method of specifically covalently labeling phosphorylated target molecules in a sample, wherein said method comprises:

• i) contacting said sample with a binding solution comprising a phosphate binding compound that is covalently bonded to a label and a photoactivatable chemically reactive group;
• a salt comprising trivalent metal ions and an acid;
• a. incubating said sample and said binding solution for sufficient time to allow said phosphate binding compound and said metal salt to associate with said phosphorylated target molecule;
• b. illuminating said phosphate binding compound with an appropriate wavelength where said photoactivatable reactive group specifically forms a covalent bond with said phosphorylated target molecule whereby said phosphorylated target molecule is labeled.

A kit for binding a phosphorylated target molecule in a sample, said kit comprising:

• i) a binding solution comprising a metal chelating moiety;
• a salt comprising trivalent metal ions, wherein said metal ion is capable of simultaneously binding said metal chelating moiety and a phosphorylated target molecule;
• and, an acid;
• ii) wherein said kit optionally includes molecular weight markers that comprise phosphorylated and non-phosphorylated polypeptides.

A method for detecting a phosphorylated target molecule in a sample wherein said sample is in an aqueous solution, wherein said method comprises:

• i) contacting said sample with a binding solution to form a combined mixture, wherein said binding solution comprises a phosphate-binding compound, a salt comprising trivalent metal ions and an acid, wherein said combined mixture comprises a first dye label that has a first absorption and emission spectra and a second dye label that has a second absorption and emission spectra;
• ii) incubating said phosphate binding compound and said sample for a sufficient amount of time for said phosphate binding compound to bind a phosphorylated target molecule;
• and,
• iii) illuminating said sample with an appropriate wavelength whereby said phosphorylated target molecule is detected by a change in fluorescence signal.

A method for detecting kinase enzymes in a sample, wherein said method comprises;

• a) contacting a sample with a kinase substrate and a source of phosphate;
• b) incubating said sample of step a) for a sufficient amount of time for said kinase substrate to be phosphorylated;
• c) contacting said phosphorylated kinase substrate with a binding solution, wherein said binding solution comprises a metal chelating moiety, a salt comprising trivalent metal ions, wherein said metal ion is capable of simultaneously binding said metal chelating moiety and a phosphorylated target molecule;
• and, an acid for a sufficient amount of time for said metal chelating moiety to bind said phosphorylated kinase substrate.
• d) illuminating said sample with an appropriate wavelength whereby said kinase substrate is detected.