A fluorescence lifetime-based assay for serine and threonine kinases that is suitable for high-throughput screening.
We describe the development of a novel method for the assay of serine/threonine protein kinases based on fluorescence lifetime. The assay consists of three generic peptides (which have been used by others in the assay of >140 protein kinases in various assay formats) labeled with a long lifetime fluorescent dye (14 or 17 ns) that act as substrates for protein kinases and an iron(III) chelate that modulates the fluorescence lifetime of the peptide only when it is phosphorylated. The decrease in average fluorescence lifetime as measured in a recently developed fluorescence lifetime plate reader (Edinburgh Instruments) is a measure of the degree of phosphorylation of the peptide. We present data showing that the assay performs as well as, and in some cases better than, the "gold standard" radiometric kinase assays with respect to Z' values, demonstrating its utility in high-throughput screening applications. We also show that the assay gives nearly identical results in trial screening to those obtained by radiometric assays and that it is less prone to interference than simple fluorescence intensity measurements.
Division of Molecular Physiology, University of Dundee, James Black Centre Dow Street, Dundee DD15EH, UK.
Michael J
MJ
Paterson
Colin J
CJ
Dunsmore
Reynald
R
Hurteaux
Beatrice A
BA
Maltman
Graham J
GJ
Cotton
Alexander
A
Gray
D000595:Amino Acid Sequence; D002614:Chelating Agents / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D057166:High-Throughput Screening Assays / Q000379:methods*; D007501:Iron / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D013379:Substrate Specificity
D002614:Chelating Agents; D005456:Fluorescent Dyes; D010455:Peptides; D007501:Iron; D017346:Protein Serine-Threonine Kinases