Hauser, Patrick and Hofmann, Francesco (2005) High-throughput assay to monitor formation of the E2-ubiquitin thioester intermediate. Methods in Enzymology, 398. pp. 87-100. ISSN 0076-6879

Abstract

Targeting components of ubiquitination pathways for drug discovery necessitates the development of high-capacity assays that monitor the ubiquitination process at defined steps of the E1-E2-E3 cascade. This chapter describes the development of an assay based on time-resolved fluorescence to monitor formation of the thioester intermediate between ubiquitin-conjugating enzymes (E2s) and ubiquitin. The methodology is exemplified by an assay tailored for the ubiquitin-conjugating enzyme Cdc34. This assay setup can be easily adapted to other E2s and is suitable to screen small molecule inhibitors of E2-thioester formation in a high-throughput mode.

Item Type:	Article
Related URLs:	http://www.ncbi.nlm.nih.gov/sites/entrez... doi:10.1016/S0076-6879(05)98009-9
Additional Information:	author can archive post-print (ie final draft post-refereeing); Publisher's version/PDF cannot be used
Related URLs:	http://www.ncbi.nlm.nih.gov/sites/entrez... doi:10.1016/S0076-6879(05)98009-9
Date Deposited:	14 Dec 2009 13:59
Last Modified:	31 Jan 2013 01:16
URI:	https://oak.novartis.com/id/eprint/482