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Analysis of Protein–Peptide Interaction by a Miniaturized Fluorescence Polarization Assay Using Cyclin-Dependent Kinase 2/Cyclin E as a Model System

1999; Elsevier BV; Volume: 275; Issue: 2 Linguagem: Inglês

10.1006/abio.1999.4331

1096-0309

Sokhom S. Pin, Ilona Kariv, Nilsa Graciani, Kevin R. Oldenburg,

14-3-3 protein interactions

As a result of the increasing size of chemical libraries, more rapid and highly sensitive strategies are needed to accelerate the process of drug discovery without increasing the cost. One means of accomplishing this is to miniaturize the assays that enter high-throughput screening (HTS). Miniaturization requires an assay design that has few steps, has a large degree of separation between the signal and background, and has a low well to well signal variation. Fluorescence polarization (FP) is an assay type that, in many cases, meets all of the above requirements. FP is a homogenous method that allows interactions between molecules to be measured directly in solution. This article demonstrates the application of FP in a miniaturized HTS format, using 1536-well plates, to measure direct binding between cyclin-dependent kinase 2/cyclin E complex (CDK2/E) and an 8-mer-peptide kinase inhibitor. The data indicate that low variability and high specificity allow rapid and precise identification of antagonist compounds affecting CDK2/E-peptide interactions.