COMPOSITIONS AND METHODS FOR DETECTION AND ISOLATION OF PHOSPHORYLATED MOLECULES
First Claim
1. A method for binding a phosphorylated target molecule in a sample, said method comprising the steps of:
- i) contacting said sample with a binding solution comprising;
a) a BAPTA metal chelating moiety;
b) a salt comprising trivalent metal ions; and
, c) an acid; and
, ii) incubating said sample and said binding solution for sufficient time to allow said phosphate-binding compound to associate with said phosphorylated target molecule whereby said phosphorylated target molecule is bound.
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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.
122 Citations
28 Claims
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1. A method for binding a phosphorylated target molecule in a sample, said method comprising the steps of:
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i) contacting said sample with a binding solution comprising;
a) a BAPTA metal chelating moiety;
b) a salt comprising trivalent metal ions; and
,c) an acid; and
,ii) incubating said sample and said binding solution for sufficient time to allow said phosphate-binding compound to associate with said phosphorylated target molecule whereby said phosphorylated target molecule is bound. - View Dependent Claims (2, 3, 4, 5)
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6. A method for detecting a phosphorylated target molecule in a sample immobilized on a membrane, said method comprising the steps of:
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i) optionally electrophoretically separating said sample on a gel;
ii) immobilizing said sample to a membrane;
iii) optionally contacting said membrane of step ii) with a fixing solution;
iv) contacting said membrane of step iii) with a binding solution, wherein said binding solution comprises;
a) a phosphate-binding compound having formula (A)m(L)n(B) wherein A is a chemical moiety, L is a linker, B is a metal-chelating moiety, m is an integer from 1 to 4 and n is an integer from 0 to 4;
b) a salt comprising metal ions; and
,c) an acid;
v) incubating said membrane of step iv) and said binding solution for sufficient time to allow said phosphate-binding compound to associate with said phosphorylated target molecule; and
,vi) visualizing said compound whereby said phosphorylated target molecule is detected. - View Dependent Claims (7, 8, 9, 10, 11, 12)
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13. A method for detecting a phosphorylated target molecule in a sample immobilized on a gel, said method comprising the steps of:
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i) immobilizing said sample on a gel;
ii) optionally contacting said gel of step i) with a fixing solution;
iii) contacting said gel of step ii) with a binding solution, wherein said binding solution comprises;
a) a compound having formula (A)m(L)n(B) wherein A is a chemical moiety, L is a linker, B is a metal-chelating moiety, m is an integer from 1 to 4 and n is an integer from 0 to 4;
b) a salt comprising metal ions; and
,c) an acid;
iv) incubating said gel of step iii) and said binding solution for sufficient time to allow said compound to associate with said phosphorylated target molecule; and
,v) visualizing said compound whereby said phosphorylated target molecule is detected. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21)
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22. A method for detecting a phosphorylated target molecule in a sample immobilized on a matrix, said method comprising the steps of:
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i) immobilizing said sample on an matrix;
ii) contacting said matrix of step i) with a binding solution, wherein said binding solution comprises;
a) a phosphate-binding compound having formula (A)m(L)n(B) wherein A is a chemical moiety, L is a linker, B is a metal-chelating moiety, m is an integer from 1 to 4 and n is an integer from 0 to 4;
b) a salt comprising metal ions; and
,c) an acid;
iii) incubating said matrix of step ii) and said binding solution for sufficient time to allow said compound to associate with said phosphorylated target molecule; and
,iv) visualizing said compound whereby said phosphorylated target molecule is detected. - View Dependent Claims (23, 24, 25, 26, 27, 28)
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Specification