Compositions and methods for detection and isolation of phosphorylated molecules
First Claim
1. A binding solution comprising:
- a) a metal chelating moiety;
b) 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
, c) an acid.
4 Assignments
0 Petitions
Accused Products
Abstract
The present invention relates to phosphate-binding compounds that find use in binding, detecting and isolating phosphorylated target molecules including the subsequent identification of target molecules that interact with phosphorylated target molecules or molecules capable of being phosphorylated. A binding solution is provide that comprises a phosphate-binding compound, an acid and a metal ion wherein the metal ion simultaneously interacts with an exposed phosphate group on a target molecule and the metal chelating moiety of the phosphate-binding compound forming a bridge between the phosphate-binding compound and a phosphorylated target molecule resulting in a ternary complex. The binding solution of the present invention finds use in binding and detecting immobilized and solubilized phosphorylated target molecules, isolation of phosphorylated target molecules from a complex mixture and aiding in proteomic analysis wherein kinase and phosphatase substrates and enzymes can be identified.
123 Citations
56 Claims
-
1. A binding solution comprising:
-
a) a metal chelating moiety;
b) 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
,c) an acid. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 43)
-
-
19. 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. - View Dependent Claims (20, 21, 22, 23, 24, 25, 26, 27, 28)
-
29. 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. - View Dependent Claims (30, 31, 32, 33, 34, 35)
-
- 36. A matrix comprising an immobilized phosphate-binding compound comprising a metal chelating moiety that is capable of specifically binding to a phosphorylated target molecule.
- 38. A phosphate-binding compound, wherein said compound comprises a metal chelating moiety and a covalently bonded photoactivatable chemically reactive group, where said metal chelating moiety is capable of specifically binding to a phosphorylated target molecule.
- 40. A ternary complex comprising a trivalent metal ion, a phosphorylated target molecule and a phosphate-binding compound comprising a metal chelating moiety wherein said metal ion is capable of simultaneously binding said phosphorylated target molecule and said metal chelating moiety.
-
44. 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. - View Dependent Claims (45, 46, 47, 48, 49, 50)
-
-
51. 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. - View Dependent Claims (52, 53, 54, 55, 56)
-
Specification