Assay methods and systems

• US 20070161071A1
• Filed: 11/10/2006
• Published: 07/12/2007
• Est. Priority Date: 05/21/1999
• Status: Abandoned Application

• Current Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Original Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Inventors: Nikiforov, Theo
• Application Number:
  US11/595,271
• Publication Number:
  US 20070161071A1
• Time in Patent Office: Days
• US Class Current: 435/15
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: G01N 2333/9121 with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching

Abstract

Methods, systems, kits for carrying out a wide variety of different assays that comprise providing a first reagent mixture which comprises a first reagent having a fluorescent label. A second reagent is introduced into the first reagent mixture to produce a second reagent mixture, where the second reagent reacts with the first reagent to produce a fluorescently labeled product having a substantially different charge than the first reagent. A polyion is introduced into at least one of the first and second reagent mixtures, and the fluorescent polarization in the second reagent mixture relative to the first reagent mixture is determined, this fluorescent polarization being indicative of the rate or extent of the reaction.

Claims

1. A method of monitoring the activity of a kinase enzyme, comprising:

providing a first mixture comprising a phosphorylatable compound that includes a fluorescent label, a kinase enzyme and a phosphate donor group, wherein the kinase enzyme is capable of phosphorylating the phosphorylatable compound to produce a phosphorylated product;

contacting the phosphorylated product with a molecule having multivalent metal cations associated therewith; and

determining a level of phosphorylated product produced by the activity of the kinase enzyme by measuring binding of the molecule to the phosphorylated product.

2. The method of claim 1, wherein the compound comprises a serine, tyrosine, or threonine substrate.

3. The method of claim 1, wherein the multivalent metal cations bind the molecule to the phosphorylated product based at least partially on a difference in charge between the phosphorylated product and the multivalent metal cations.

4. The method of claim 1, wherein the multivalent metal cations bind the molecule to the phosphorylated product based at least partially on an affinity between the metal cations and a phosphate group associated with the phosphorylated product

5. The method of claim 1, wherein the multivalent metal cations comprise trivalent metal cations.

6. The method of claim 5, wherein the trivalent metal cations comprise Fe³⁺.