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Biological Procedures Online

, Volume 1, Issue 1, pp 92–99 | Cite as

A method for assaying deubiquitinating enzymes

  • Jae Il Lee
  • Seung Kyoon Woo
  • Keun Il Kim
  • Kyung Chan Park
  • Sung Hee Baek
  • Yung Joon Yoo
  • Chin Ha Chung
Open Access
Article

Abstract

A general method for the assay of deubiquitinating enzymes was described in detail using 125I-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.

Keywords

Assay Protocol Biological Procedure Strain AR13 Deubiquitinating Enzyme Biological Procedure Online 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
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Copyright information

© Springer 1998

Authors and Affiliations

  • Jae Il Lee
    • 1
  • Seung Kyoon Woo
    • 1
  • Keun Il Kim
    • 1
  • Kyung Chan Park
    • 1
  • Sung Hee Baek
    • 1
  • Yung Joon Yoo
    • 2
    • 1
  • Chin Ha Chung
    • 1
  1. 1.Department of Molecular Biology and Research Center for Cell Differentiation, College of Natural SciencesSeoul National UniversitySeoulKorea
  2. 2.Department of Life ScienceKwangju Institute of Science and TechnologyKwangjuKorea

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