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

TitleQuantification of protein kinase enzymatic activity in unfractionated cell lysates using CSox-based sensors.
Publication TypeJournal Article
Year of Publication2014
AuthorsBeck, JR, Peterson, LB, Imperiali, B, Stains, CI
JournalCurr Protoc Chem Biol
Volume6
Issue3
Pagination135-56
Date Published2014
ISSN2160-4762
KeywordsAmino Acids, Animals, Biosensing Techniques, Cell-Free System, Cells, Cultured, Fluorescence, Molecular Probes, Phosphorylation, Protein Kinases
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.

DOI10.1002/9780470559277.ch140106
Alternate JournalCurr Protoc Chem Biol
PubMed ID25205563
PubMed Central IDPMC4174361
Grant ListF32 GM102992 / GM / NIGMS NIH HHS / United States
F32 GM102992 / GM / NIGMS NIH HHS / United States
U54 CA112967 / CA / NCI NIH HHS / United States
U54-CA112967 / CA / NCI NIH HHS / United States