Title | Quantification of protein kinase enzymatic activity in unfractionated cell lysates using CSox-based sensors. |
Publication Type | Journal Article |
Year of Publication | 2014 |
Authors | Beck, JR, Peterson, LB, Imperiali, B, Stains, CI |
Journal | Curr Protoc Chem Biol |
Volume | 6 |
Issue | 3 |
Pagination | 135-56 |
Date Published | 2014 |
ISSN | 2160-4762 |
Keywords | Amino 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. |
DOI | 10.1002/9780470559277.ch140106 |
Alternate Journal | Curr Protoc Chem Biol |
PubMed ID | 25205563 |
PubMed Central ID | PMC4174361 |
Grant List | F32 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 |