Cell-line-specific high background in the Proteasome-Glo assay of proteasome trypsin-like activity.
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.
Department of Pharmacology and Toxicology, Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, NH 03756, USA.
Owen M
OM
Wilkins
Department of Pharmacology and Toxicology, Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, NH 03756, USA.
Sondra L
SL
Downey
Department of Pharmacology and Toxicology, Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, NH 03756, USA.
Emily S
ES
Weyburne
Department of Pharmacology and Toxicology, Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, NH 03756, USA.
David A
DA
Williams
Department of Pharmacology and Toxicology, Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, NH 03756, USA.
Anne C
AC
Mirabella
Leiden Institute of Chemistry and Netherlands Proteomics Centre, Gorlaeus Laboratories, 2333 CC Leiden, The Netherlands.
Herman S
HS
Overkleeft
Department of Pharmacology and Toxicology, Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, NH 03756, USA. Electronic address: alexei.f.kisselev@dartmouth.edu.
Alexei F
AF
Kisselev
D045744:Cell Line, Tumor; D005456:Fluorescent Dyes / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008163:Luminescent Measurements; D046988:Proteasome Endopeptidase Complex / Q000032:analysis* / Q000378:metabolism; D013379:Substrate Specificity; D014357:Trypsin / Q000032:analysis / Q000378:metabolism
D005456:Fluorescent Dyes; D014357:Trypsin; D046988:Proteasome Endopeptidase Complex
Protease; Proteasome; Ubiquitin