Assay for detecting the enzymatic activity of a phosphorylation enzyme using enhanced signal generation
AssigneeKinetek Pharmaceuticals, Inc.
InventorsZaihui Zhang, Jasbinder Sanghera, Shisen Wang
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Abstract
An assay system is provided for detecting the enzymatic activity of a phosphorylation enzyme. The enzyme may be a phosphatase or protein kinase. The substrate for the enzyme is immobilized on a solid support via covalent or non-covalent binding through a signal enhancing polymer. The immobilized substrate provides an enhanced signal to background ratio when compared to a substrate in solution. The methods are easily adapted to high throughput screening systems.
Claims25
An improved method for screening samples to perform a phosphorylation assay, wherein the sensitivity of said assay is improved relative to an unconjugated peptide substrate by the method comprising:
- conjugating a peptide substrate to a signal enhancing polymer;
- immobilizing said polymer and conjugated peptide substrate on a solid surface;
- exposing said immobilized substrate to a mixture comprising a candidate modulating agent for phosphorylation;
- a phosphorylation enzyme, and detectably labeled source of phosphate groups, for a period of time sufficient for said enzyme to act on said substrate, and
- measuring the degree of phosphorylation of the substrate.
An improved method for affinity purification of phosphorylation enzymes, wherein the sensitivity is improved by the method comprising:
- conjugating a peptide substrate to a signal enhancing polymer;
- immobilizing said polymer and conjugated peptide substrate on a matrix;
- exposing said immobilized substrate to a complex mixture comprising a candidate phosphorylation enzyme and detectably labeled source of phosphate groups;
- for a period of time sufficient for said enzyme to bind to said substrate;
- washing said matrix free of unbound material;
- and
- eluting said candidate phosphorylation enzyme from said matrix.
Dependencies are inferred from the claim text.