title: Crude and purified proteasome activity assays are affected by type of microplate.
abstract: Measurement of proteasome activity is fast becoming a commonly used assay in many laboratories. The most common method to measure proteasome activity involves measuring the release of fluorescent tags from peptide substrates in black microplates. Comparisons of black plates used for measuring fluorescence with different properties show that the microplate properties significantly affect the measured activities of the proteasome. The microplate that gave the highest reading of trypsin-like activity of the purified 20S proteasome gave the lowest reading of chymotrypsin-like activity of the 20S proteasome. Plates with medium binding surfaces from two different companies showed an approximately 2-fold difference in caspase-like activity for purified 20S proteasomes. Even standard curves generated using free 7-amino-4-methylcoumarin (AMC) were affected by the microplate used. As such, significantly different proteasome activities, as measured in nmol AMC released/mg/min, were obtained for purified 20S proteasomes as well as crude heart and liver samples when using different microplates. The naturally occurring molecule betulinic acid activated the chymotrypsin-like proteasome activity in three different plates but did not affect the proteasome activity in the nonbinding surface microplate. These findings suggest that the type of proteasome activity being measured and sample type are important when selecting a microplate.
authors.affiliation: Department of Neurobiology, Physiology, and Behavior, University of California, Davis, CA 95616, USA.
authors.forename: Ziyou
authors.initials: Z
authors.lastname: Cui
authors.affiliation: Department of Neurobiology, Physiology, and Behavior, University of California, Davis, CA 95616, USA.
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Gilda
authors.affiliation: Department of Neurobiology, Physiology, and Behavior, University of California, Davis, CA 95616, USA; Department of Physiology and Membrane Biology, University of California, Davis, CA 95616, USA. Electronic address: avgomes@ucdavis.edu.
authors.forename: Aldrin V
authors.initials: AV
authors.lastname: Gomes
mesh_terms: D000818:Animals; D002138:Calibration; D057075:Enzyme Assays / Q000379:methods*; D009928:Organ Specificity; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D040081:Protein Array Analysis / Q000379:methods*; D051381:Rats
chemical_list: D046988:Proteasome Endopeptidase Complex
keywords: Betulinic acid; Microplate; Proteasome; Proteolytic activity