title: Fluorescence-based proteasome activity profiling.
abstract: With the proteasome emerging as a therapeutic target for cancer treatment, accurate tools for monitoring proteasome (inhibitor) activity are in demand. In this chapter, we describe the synthesis and use of a fluorescent proteasome activity probe that allows for accurate profiling of proteasomal activity in cell lysates, intact cells, and murine and human patient-derived material, with high sensitivity using SDS-PAGE. The probe allows for direct scanning of the gel for fluorescent emission of the distinct proteasomal subunits and circumvents the use of Western blot analysis. Due to its suitable biochemical and biophysical properties, the fluorescent probe can also be used for confocal laser scanning microscopy and flow cytometry-based experiments.
authors: Division of Cell Biology II, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
authors: Annemieke
authors: A
authors: de Jong
authors: Karianne G
authors: KG
authors: Schuurman
authors: Boris
authors: B
authors: Rodenko
authors: Huib
authors: H
authors: Ovaa
authors: Celia R
authors: CR
authors: Berkers
mesh_terms: D000818:Animals; D001671:Biochemistry / Q000379:methods*; D001681:Biological Assay; D001896:Boron Compounds / Q000138:chemical synthesis / Q000737:chemistry; D001897:Boronic Acids / Q000737:chemistry / Q000494:pharmacology; D000069286:Bortezomib; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D056148:Chromatography, Reverse-Phase; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D051379:Mice; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism; D011719:Pyrazines / Q000737:chemistry / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D001897:Boronic Acids; D005456:Fluorescent Dyes; D021122:Protein Subunits; D011719:Pyrazines; D000069286:Bortezomib; D046988:Proteasome Endopeptidase Complex