Quantification of protein kinase enzymatic activity in unfractionated cell lysates using CSox-based sensors

Jon R. Beck, Laura B. Peterson, Barbara Imperiali, Clifford I Stains

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

Defining perturbations in protein kinase activity within biological samples can provide insight into disease mechanisms as well as potential targets for drug development. In this article, we present a method that utilizes a phosphorylation-sensitive amino acid, termed CSox, to afford kinase-selective biosensors capable of reporting on enzymatic activity directly in biological samples. These sensors produce an increase in fluorescence in response to phosphorylation of an amino acid residue adjacent to CSox. Probes can be designed for either serine/threonine or tyrosine kinases, and analysis can be performed using standard fluorescence equipment. The procedures provided herein represent our optimized protocols for the design, validation, and application of CSox-based protein kinase activity sensors.

Original languageEnglish (US)
Pages (from-to)135-156
Number of pages22
JournalCurrent protocols in chemical biology
Volume6
Issue number3
DOIs
Publication statusPublished - Jan 1 2014

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Keywords

  • cell signaling
  • fluorescence-based biosensor
  • kinase activity assay
  • kinase activity profiling
  • phosphorylation

ASJC Scopus subject areas

  • Medicine(all)

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