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Biological Procedures Online

, Volume 1, Issue 1, pp 92–99

DOI: 10.1251-bpo11

Cite this article as: Lee, J.I., Woo, S.K., Kim, K.I. et al. Biol Proced Online 1998 1: 92. doi:10.1251-bpo11

Abstract

A general method for the assay of deubiquitinating enzymes was described in detail using I-labeled ubiquitin-fused αNH-MHISPPEPESEEEEEHYC referred to as Ub-PESTc as a substrate. Since the tyrosine residue in the PESTc portion of the fusion protein was almost exclusively radioiodinated under a mild labeling condition, such as using IODO-BEADS, the enzymes could be assayed directly by simple measurement of the radioactivity released into acid soluble products. Using this assay protocol, we could purify six deubiquitinating enzymes from chick skeletal muscle and yeast and compare their specific activities. Since the extracts of E. coli showed little or no activity against the substrate, the assay protocol should be useful for identification and purification of eukaryotic deubiquitinating enzymes cloned and expressed in the cells.

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Autor: Jae Il Lee - Seung Kyoon Woo - Keun Il Kim - Kyung Chan Park - Sung Hee Baek - Yung Joon Yoo - Chin Ha Chung

Fuente: https://link.springer.com/







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