title: Fetal calf sera can distort cell-based luminescent proteasome assays through heat-resistant chymotrypsin-like activity.
abstract: Luminescence-based proteasome activity assays use specific substrates that are supposed to be cleaved by cellular proteasome activity leading to luciferase substrates. Usually, control wells containing cell culture medium supplemented with antibiotics and fetal calf serum are used as background. Using the Proteasome-Glo chymotrypsin-like cell-based assay from Promega, we show here that fetal calf sera from different manufacturers contain heat-resistant, bortezomib-inhibitable, chymotrypsin-like activities that can interfere with proteasome activity assays. These data strongly recommend the use of pure phosphate-buffered saline (PBS) or serum-free medium during proteasome activity assays to diminish background luminescence and, thus, to obtain reliable results.
authors.affiliation: Department of Clinical Pharmacology and Pharmacoepidemiology, Heidelberg University Hospital, D-69120 Heidelberg, Germany.
authors.forename: Susan
authors.initials: S
authors.lastname: Dettmer
authors.affiliation: Department of Clinical Pharmacology and Pharmacoepidemiology, Heidelberg University Hospital, D-69120 Heidelberg, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Theile
authors.affiliation: Department of Internal Medicine V, Oncology, Hematology, and Rheumatology, Heidelberg University Hospital, D-69120 Heidelberg, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Seckinger
authors.affiliation: Department of Clinical Pharmacology and Pharmacoepidemiology, Heidelberg University Hospital, D-69120 Heidelberg, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Burhenne
authors.affiliation: Department of Clinical Pharmacology and Pharmacoepidemiology, Heidelberg University Hospital, D-69120 Heidelberg, Germany. Electronic address: johanna.weiss@med.uni-heidelberg.de.
authors.forename: Johanna
authors.initials: J
authors.lastname: Weiss
mesh_terms: D000818:Animals; D016477:Artifacts; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D008163:Luminescent Measurements; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D044967:Serum / Q000378:metabolism*
chemical_list: D002918:Chymotrypsin; D046988:Proteasome Endopeptidase Complex
keywords: Bortezomib; Carfilzomib; Fetal calf serum; Heat resistance; Proteasome activity; Proteasome-Glo chymotrypsin-like cell-based assays