title: Quantification of protein kinase enzymatic activity in unfractionated cell lysates using CSox-based sensors.
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.
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska.
authors.forename: Jon R
authors.initials: JR
authors.lastname: Beck
authors.affiliation: Department of Biology and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts.
authors.forename: Laura B
authors.initials: LB
authors.lastname: Peterson
authors.affiliation: Department of Biology and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts.
authors.forename: Barbara
authors.initials: B
authors.lastname: Imperiali
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska.
authors.forename: Cliff I
authors.initials: CI
authors.lastname: Stains
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002474:Cell-Free System; D002478:Cells, Cultured; D005453:Fluorescence; D015335:Molecular Probes / Q000737:chemistry; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D015335:Molecular Probes; D011494:Protein Kinases
keywords: cell signaling; fluorescence-based biosensor; kinase activity assay; kinase activity profiling; phosphorylation