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US 9,284,598 B2Granted
Methods to assay phosphatase activity
AssigneeFluidigm Canada Inc.
InventorOlga Ornatsky
Life of this patent
Estimating the term…
Abstract
Methods and kits for enzymes involved in post-translational modifications are provided. The methods employ elemental analysis, including ICP-MS. The methods allow for the convenient and accurate analysis of post-translation modifications of substrates by enzymes involved in post-translational modifications, including kinase and phosphatase enyzmes.
Claims14
A method for a phosphatase assay, comprising:
- a) incubating free phosphorylated substrates labeled with element tags with a support having attached thereto metal ion coordination complexes such that at least some of the free phosphorylated substrates bind with the metal ion coordinate complexes attached to the support to form bound phosphorylated substrates;
- b) separating the remaining free phosphorylated substrates from the bound phosphorylated substrates;
- c) incubating ADP and at least one phosphatase with the bound phosphorylated substrates under conditions to enable the phosphatase to dephosphorylate the bound phosphorylated substrates to form free non-phosphorylated substrates;
- d) separating the free non-phosphorylated substrates from the remaining bound phosphorylated substrates;
- and
- e) measuring at least one of a phosphatase activity, specificity, and concentration using elemental analysis to detect the element tags associated with the free non-phosphorylated substrates.
3 more elements
A method for a phosphatase assay, comprising:
- a) incubating in a plurality of solutions a plurality of element labeled supports, the plurality of element labeled supports each having attached thereto a metal ion coordination complex, wherein the plurality of solutions comprise different solutions, and wherein each solution includes a different type of free phosphorylated substrate labeled with an element tag, wherein after incubation, at least a portion of the free phosphorylated substrate attaches to the metal ion coordinate complex of the element labeled support to form bound phosphorylated substrates, and wherein each type of bound phosphorylated substrate is attached to a single type of element labeled support;
- b) separating, after incubation, remaining free phosphorylated substrates from the bound phosphorylated substrates;
- c) incubating the bound phosphorylated substrates with ADP and at least one phosphatase in conditions that enable the phosphatase to dephosphorylate at least a portion of the bound phosphorylated substrates to form free non-phosphorylated substrates;
- d) separating free non-phosphorylated substrates from remaining bound phosphorylated substrates;
- and
- e) measuring at least one of phosphatase activity, specificity, and concentration by performing particle elemental analysis of remaining bound phosphorylated substrates.
3 more elements
A method for a phosphatase assay, comprising:
- a) incubating ADP and at least one phosphatase with immobilized phosphorylated substrates with attached metal ion coordination complexes in conditions that enable the phosphatase to dephosphorylate the phosphorylated substrates to form immobilized non-phosphorylated substrates and free metal ion coordination complexes;
- b) separating the free metal ion coordination complexes from the immobilized non-phosphorylated substrates and the immobilized phosphorylated substrates with attached metal ion coordination complexes;
- c) eluting the free metal ion coordination complexes into a solution;
- and
- d) measuring at least one of phosphatase activity, specificity, and concentration by measuring the solution by elemental analysis.
2 more elements
A method for phosphatase assay, comprising:
- a) incubating ADP, at least one phosphatase, and a plurality of types of phosphorylated substrates with attached metal ion coordination complexes immobilized to element labeled supports, wherein a single type of phosphorylated substrate is attached to a single type of element labeled support;
- b) separating free metal ion coordination complexes formed by dephosphorylation of the phosphorylated substrates by the phosphatase from non-phosphorylated substrates immobilized to element labeled supports and immobilized phosphorylated substrates;
- and
- c) measuring at least one of phosphatase activity, specificity, and concentration by measuring the metal ion coordination complexes attached to the phosphorylated substrates immobilized to element labeled supports by elemental analysis.
1 more element
Dependencies are inferred from the claim text.