Development, validation and implementation of immobilized metal affinity for phosphochemicals (IMAP)-based high-throughput screening assays for low-molecular-weight compound libraries.
This protocol describes assay development, validation and implementation of automated immobilized metal affinity for phosphochemicals (IMAP)-based fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET) high-throughput screening (HTS) assays for identification of low-molecular-weight kinase inhibitors. Both procedures are performed in miniaturized kinase reaction volumes and involve the stepwise addition of test or control compounds, enzyme and substrate/ATP. Kinase reactions are stopped by subsequent addition of IMAP-binding buffer. Assay attributes of the IMAP FP and TR-FRET methodologies are described. HTS assays developed using these procedures should result in Z-factors and low assay variability necessary for robust HTS assays. Providing that the required reagents and equipment are available, one scientist should be able to develop a 384-well, miniaturized HTS assay in approximately 6-8 weeks. Specific automated HTS assay conditions will determine the number of assay plates processed in a screening session, but two scientists should expect to process between 100 and 150 assay plates in one 8-h screening day.
Sharlow
Elizabeth R
ER
Drug Discovery Institute, Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA. ers@pitt.edu
Leimgruber
Stephanie
S
Yellow-Duke
Archibong
A
Barrett
Rebecca
R
Wang
Qiming Jane
QJ
Lazo
John S
JS
D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors; D004795:Enzyme Stability; D005454:Fluorescence Polarization / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D010770:Phosphotransferases / Q000737:chemistry*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D047428:Protein Kinase Inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors; D054852:Small Molecule Libraries; D000097869:Polo-Like Kinase 1
D018797:Cell Cycle Proteins; D010770:Phosphotransferases; D011493:Protein Kinase C; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D054852:Small Molecule Libraries; D000097869:Polo-Like Kinase 1; C089212:protein kinase D