An improved zinc cocktail-mediated fluorescence polarization-based kinase assay for high-throughput screening of kinase inhibitors.
During the past few years, high-throughput screening (HTS) has provided a useful resource to researchers involved in the development of kinase inhibitors as a novel therapeutic modality. However, with all the choices among kinase assays, there is not yet a one-size-fits-all assay. Therefore, selection of a specific kinase assay is a daunting task. HTS assays should be homogeneous, cost effective, use nonradioactive reagents, generic and not time consuming. Here, we report an improved method of assaying protein kinase activity using a zinc cocktail in a fluorescence polarization-(FP) based format. Assay conditions were standardized manually and validated in a HTS format using a liquid handler. We validated this assay for both serine/threonine and tyrosine (receptor/nonreceptor) kinases. The results obtained in the HTS assay system were comparable to the commercially available fluorescence-based assay. We suggest that the reported assay is a cost-effective alternative to the IMAP-based generic kinase assay.
Department of Pharmacology, New Drug Discovery Research, Ranbaxy Research Laboratories, Plot No-20, Sector-18, Gurgaon, 122001 Haryana, India. p.chopra@ranbaxy.com
Puneet
P
Chopra
Kamna
K
Nanda
Mou
M
Chatterjee
Malini
M
Bajpai
Sunanda G
SG
Dastidar
Abhijit
A
Ray
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002712:Chlorides / Q000737:chemistry* / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005454:Fluorescence Polarization / Q000379:methods*; D020128:Inhibitory Concentration 50; D008969:Molecular Sequence Data; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D017967:Zinc Compounds / Q000737:chemistry* / Q000378:metabolism*
D002712:Chlorides; D047428:Protein Kinase Inhibitors; D017967:Zinc Compounds; C016837:zinc chloride; D011494:Protein Kinases