期刊
NATURE PROTOCOLS
卷 3, 期 8, 页码 1350-1363出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2008.111
关键词
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资金
- National Institutes of Health [R03 DA24898-01, U54 MH074411-03, X01 MH077611-01]
- NATIONAL CANCER INSTITUTE [R01CA142580] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF MENTAL HEALTH [U54MH074411] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE ON DRUG ABUSE [R03DA024898] Funding Source: NIH RePORTER
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 similar to 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.
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