Curtis, Ashton J; Dowsell, Ryan S; Gold, Matthew G; (2022) Assaying Protein Kinase A Activity Using a FRET-Based Sensor Purified from Mammalian Cells. Methods in Molecular Biology , 2483 pp. 15-31. 10.1007/978-1-0716-2245-2_2.

Preview

Text MMB_Gold_forUCL_RPSx.pdf - Accepted Version Download (9MB) | Preview

Abstract

Protein Kinase A (PKA) is the major intracellular receptor for cAMP. Research into this prototype kinase is supported by kinase assays that are typically performed in vitro using radio-labeled ATP. For in vivo studies, genetically encoded FRET-based sensors have become popular for monitoring PKA activity. Here, we show that it is also possible to apply such reporters in vitro. We describe how to express and purify milligram quantities of a FRET-based PKA activity reporter using cultured human embryonic kidney cells. We demonstrate how to utilize the purified reporter in a plate reader to determine the IC50 for the widely utilized PKA inhibitor H89 in the presence of a physiologically relevant concentration of ATP. The protocol takes advantage of the economical transfection reagent polyethylenimine and can be performed in a standard cell culture facility. Whereas assays based on radiolabelling are more sensitive, the approach presented here has several advantages: It enables continuous measurement of changes in substrate phosphorylation; a single preparation produces enough reporter for thousands of recordings; the reporter has a long shelf life; and it avoids the safety considerations that arise when working with radioactive material.

Download activity - last month

Export as CSVXMLJSON

Download activity - last 12 months

Export as XMLJSONCSV

Downloads by country - last 12 months

Export as CSVXMLJSON

Archive Staff Only

View Item