期刊
ANALYTICAL BIOCHEMISTRY
卷 482, 期 -, 页码 7-15出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2015.04.019
关键词
Proteasome; Activity assay; Fluorogenic peptides; Proteasome-Glo
资金
- Belgian Program on Interuniversity Poles of Attraction initiated by the Belgian State (Prime Minister's Office, Science Policy Programming)
- Fonds National de la Recherche Scientifique (Fonds de la Recherche Scientifique - FNRS, Belgium)
- Fondation contre le Cancer (a nonprofit organization in Belgium)
- Fonds J. Maisin (Belgium)
- Fondation Salus Sanguinis (Belgium)
- Ludwig Institute for Cancer Research and WELBIO
Because of its crucial role in various cellular processes, the proteasome is the focus of intensive research for the development of proteasome inhibitors to treat cancer and autoimmune diseases. Here, we describe a new and easy assay to measure the different proteasome activities in vitro (chymotrypsin-like, caspase-like, and trypsin-like) based on proteasome capture on antibody-coated plates, namely the capture proteasome assay (CAPA). Applying the CAPA to lysates from cells expressing standard proteasome, immunoproteasome, or intermediate proteasomes beta 5i or beta 1i-beta 5i, we can monitor the activity of the four proteasome subtypes. The CAPA provided similar results as the standard whole-cell proteasome-Glo assay without the problem of contaminating proteases requiring inhibitors. However, the profile of trypsin-like activity differed between the two assays. This could be partly explained by the presence of MgSO4 in the proteasome-Glo buffer, which inhibits the tiypsin-like activity of the proteasome. The CAPA does not need MgSO4 and, therefore, provides a more precise measurement of the trypsin-like activity. The CAPA provides a quick and accurate method to measure proteasome activity in vitro in a very specific manner and should be useful for the development of proteasome inhibitors. (C) 2015 Elsevier Inc. All rights reserved.
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