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Detection of p56lck Kinase Activity Using Scintillation Proximity Assay in 384-Well Format and Imaging Proximity Assay in 384- and 1536-Well Format

Amersham Pharmacia Biotech UK Limited, Little Chalfont, Buckinghamshire, UK

Young-Whan Park

Merck Research Laboratories, Rahway, NJ

Jeff Hermes

Merck Research Laboratories, Rahway, NJ

Angela Marenghi

Amersham Pharmacia Biotech UK Limited, Little Chalfont, Buckinghamshire, UK

Gerard Brophy

Amersham Pharmacia Biotech UK Limited, Little Chalfont, Buckinghamshire, UK

Albert Santos

Amersham Pharmacia Biotech UK Limited, Little Chalfont, Buckinghamshire, UK

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 -phosphate group from [-³³P]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 LEAD-seeker^TM, 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.

Journal of Biomolecular Screening, Vol. 5, No. 4, 205-211 (2000)
DOI: 10.1177/108705710000500403


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