Cell-line-specific high background in the Proteasome-Glo assay of proteasome trypsin-like activity |
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Authors: | Owen M Wilkins Sondra L Downey Emily S Weyburne David A Williams Anne C Mirabella Herman S Overkleeft Alexei F Kisselev |
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Institution: | 1. Department of Pharmacology and Toxicology, Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, NH 03756, USA;2. Leiden Institute of Chemistry and Netherlands Proteomics Centre, Gorlaeus Laboratories, 2333 CC Leiden, The Netherlands |
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Abstract: | Proteasome-Glo is a homogeneous cell-based assay of proteasomal chymotrypsin-like, trypsin-like, and caspase-like activities using luminogenic substrates, commercially available from Promega. Here we report that the background activity from cleavage of the substrate of the trypsin-like sites by nonproteasomal proteases in multiple breast and lung cancer cell lines exceeds the activity of the proteasome. We also observed substantial background chymotrypsin-like activity in some cell lines. Thus, Proteasome-Glo assay must be used with caution, and it is necessary to include a specific proteasome inhibitor to determine the background for each proteasome activity. |
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Keywords: | Proteasome Ubiquitin Protease |
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