Detection of p56(lck) kinase activity using scintillation proximity assay in 384-well format and imaging proximity assay in 384- and 1536-well format.
p56(lck) is a lymphocyte-specific tyrosine kinase that plays an important role in both T-cell maturation and activation. We have developed a homogeneous assay in which p56(lck) catalyzes the transfer of the gamma-phosphate group from [gamma-(33)P]ATP to a biotinylated peptide substrate. The labeled peptide is then captured on a streptavidin-coated scintillation proximity assay (SPA) bead or imaging proximity bead. The SPA is counted in a microplate scintillation counter and the imaging proximity assay is counted in a charge-coupled device-based imaging system called LEADseekertrade mark, recently launched as a homogeneous imaging system by Amersham Pharmacia Biotech. We show, via time-dependence assays and inhibitor studies, that this assay can be performed in 1536-well microplate format using imaging proximity as the method of detection. The results compare favorably with the same assay performed in 384-well microplate format using both SPA and imaging proximity as the detection methods. From this study, we conclude that a kinase assay can be performed in 384- and 1536-well format using imaging as the detection method, with significant time savings over standard scintillation counting. In addition, we show cost saving advantages of 1536- over 384-well format in terms of reagent usage, higher throughput, and waste disposal.
Beveridge
M
M
Amersham Pharmacia Biotech UK Limited, Little Chalfont, Buckinghamshire, UK.
Park
Y W
YW
Hermes
J
J
Marenghi
A
A
Brophy
G
G
Santos
A
A
D000595:Amino Acid Sequence; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000032:analysis* / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D012588:Scintillation Counting / Q000295:instrumentation / Q000379:methods*; D013379:Substrate Specificity; D013601:T-Lymphocytes / Q000201:enzymology / Q000276:immunology
D004791:Enzyme Inhibitors; D010455:Peptides; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)