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A Scintillation Proximity Assay for the Raf/MEK/ERK Kinase Cascade: High-Throughput Screening and Identification of Selective Enzyme Inhibitors

A Scintillation Proximity Assay for the Raf/MEK/ERK Kinase Cascade: High-Throughput Screening and Identification of Selective Enzyme Inhibitors

O B McDonald, W J Chen, B Ellis, C Hoffman, L Overton, M Rink, A Smith, C J Marshall, E R Wood

Anal Biochem. 1999 Mar 15;268(2):318-29.

PMID: 10075822

Abstract:

We have developed a quantitative scintillation proximity assay (SPA) that reproduces the Raf/MEK/ERK signal transduction pathway. The components of this assay include human cRaf1, MEK1, and ERK2 and a biotinylated peptide substrate for ERK2. cRaf1 was expressed as a his-tagged protein in insect cells in an active form. MEK1 and ERK2 were expressed in Escherichia coli as glutathione S-transferase (GST)-fusion proteins in their inactive forms. ERK2 was removed from the GST portion of the fusion protein by cleavage with thrombin protease. When the purified components are incubated together, cRaf-1 phosphorylates and activates MEK1, MEK1 phosphorylates and activates ERK2, and ERK2 phosphorylates the peptide, biotin-AAATGPLSPGPFA. Phosphorylation of the peptide using [gamma-33P]ATP is detected following binding to streptavidin-coated SPA beads. The assay detects inhibitors of cRaf1, MEK1, or ERK2, and has been used to screen large numbers of compounds. The specific target of inhibition was subsequently identified with secondary assays described herein.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42411897	MEK1, active, GST tagged human			Price
IAR4248112	ERK2, active, GST tagged human			Price

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