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

Abstract

p56lck 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 p56lck catalyzes the transfer of the γ-phosphate group from [γ-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™, 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.

Recent Advances in Technologies for Analyzing Protein Kinases

“…33 P]ATP, it is applicable to highthroughput assays because there is no need for separation of phosphorylated peptides from the reaction mixture (18). Besides these, some other techniques such as PepTag ® assay (Promega), SignaTECT ® protein kinase assay systems (Promega) (19), SignalScout TM kinase profiling system (Stratagene, La Jolla, CA, USA), and AUSA ® universal protein kinase assay kit (Transbio Corporation, Baltimore, MD, USA) are also commercially available for protein kinase assay.…”

Automated High Throughput Screening for Serine Kinase Inhibitors Using a LEADSeeker™ Scintillation Proximity Assay in the 1536-Well Format

“…A telecentric lens is used to remove parallax errors by collecting light equally from all wells without shading. 6 Using a stacker carousel, both plate types can be loaded and measured automatically.…”

“…So far, highthroughput screening in the 1536-well format has been shown for assays based on cell suspensions 1,2 and for assays in solution based on fluorescence polarization 3,4 or fluorescence intensity. 5 Further, Beveridge and coworkers reported the detection of imaging bead scintillation in the 1536-well format by means of the LEADseeker™ Homogeneous Imaging System (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK), 6 while the task of low-volume pipetting for a bead-based assay in the 1536-well format has not yet been addressed. In an attempt to open up the applicability of this plate format to bead-based assays, we established a completely automated robust assay in the 1536-well format based on the scintillation proximity technique.…”

“…Although Beveridge and coworkers 6 have already reported the possibility of detecting scintillation in the 1536-well format, they did not address the problem of automated liquid handling of low volumes: They prepared the complete kinase assays as a large-volume premix of assay reagents and manually transferred aliquots of the mixture into the 1536well plate. In an extension of their efforts, we showed the possibility of setting up the complete bead-based assay within a 1536-well plate using a pipetting robot.…”

Miniaturization of a Hepatitis C Virus RNA Polymerase Assay Using a −102°C Cooled CCD Camera-Based Imaging System

“…LEADseeker, a CCD-based detection system (7,8), emerged recently as a sensitive and rapid instrument for measuring radioisotope activity in a field format and thus capable of handling plate formats having 96-, 384-, or 1536-wells/plate. The CCD chip is cooled to Ϫ102°C to greatly reduce dark current noise arising from thermal fluctuations at higher temperatures, while software algorithms correct for cosmic events that can create "false" signals.…”