Compositions and methods for detection and isolation of phosphorylated molecules
First Claim
1. A binding solution comprising:
- a) a BAPTA metal chelating moiety;
b) a salt comprising trivalent metal ions; and
, c) an acid.
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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.
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Citations
74 Claims
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1. A binding solution comprising:
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a) a BAPTA metal chelating moiety;
b) a salt comprising trivalent metal ions; and
,c) an acid. - View Dependent Claims (20, 21, 22, 23, 24, 48, 49, 50, 51, 52, 53, 54, 62, 63, 64, 65, 66, 67, 74)
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2. A binding solution comprising:
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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 trivalent metal ions; and
,c) an acid. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47)
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55. A method for isolating phosphorylated target molecules from a sample, said method comprising the steps of:
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i) charging a matrix comprising a metal-chelating moiety selected from the group consisting of Formula IV with a salt comprising trivalent metal ions;
ii) equilibrating said matrix with an acidic binding buffer, iii) adding said sample to said matrix wherein said phosphorylated target molecules are bound to said matrix of step ii);
iv) eluting said phosphorylated target molecules from said matrix with a base solution whereby said phosphorylated target molecules are isolated. - View Dependent Claims (56, 57, 58, 59, 60, 61)
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68. A matrix comprising an immobilized phosphate-binding compound according to Formula (A)m(L)n(B) wherein A is a chemical moiety, L is a linker, B is a metal chelating moiety according to Formula IV, m is an interger from 1 to 4 and n is an interger from 0 to 4.
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69. A compound having formula (A)m(L)n(B) wherein A is a label, L is a linker, m is an integer from 1 to 4, n is an integer from 0 to 4 and B is a metal-chelating moiety comprising Formula IV wherein said compounds are selected from the group consisting of
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70. A compound comprising a metal-chelating moiety according to Formula IV that is
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71. A compound comprising a metal-chelating moiety according to Formula IV
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72. A ternary complex comprising a trivalent gallium ion, a phosphorylated target molecule and a phosphate-binding compound according to 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 interger from 1 to 4 and n is an interger from 0 to 4.
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73. A ternary complex comprising a BAPTA chelating moiety, a trivalent gallium ion and a phosphorylated target molecule.
Specification