Assay of protein kinases with peptide substrates

US5141852A · kind A · utility

Assignee

• The United States of America, as represented by the Department of Health and Human Services · US

Inventors

• John J. Egan · New York, US
• Constantine Londos · Garrett Park, US

Key dates

• Filing date: Dec 12, 1988
• Grant date: Aug 25, 1992
• Expiry date: Dec 12, 2008

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12Q1/48
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

A method for assaying protein kinases that phosphorylate peptides such as Kemptide, such as cAMP-dependent protein kinase, or a glycogen synthase peptide, which is an excellent substrate for protein kinase C. Upon sequentially processing of reaction mixtures through tandem columns of cation and anion exchange resins improved separation of ATP from phosphorylated peptides is achieved such that radioactivity in background samples is nearly nil and the yield of phosphorylated peptides is high. This method is generally applicable to any protein kinase so long as the substrate peptide is appropriately structured such that the peptide retains a net positive charge when fully phosphorylated so that the peptide will adhere to the cation exchange resin and pass through the anion exchange resin. This method reduces labor, radioactivity, enzyme requirements, and costs of assaying protein kinases.