Detection of Active Caspases During Apoptosis Using Fluorescent Activity-Based Probes.
Activity-based probes (ABPs) are reactive small molecules that covalently bind to active enzymes. When tagged with a fluorophore, ABPs serve as powerful tools to investigate enzymatic activity across a wide variety of applications. In this chapter, we provide detailed methods for using fluorescent ABPs to detect the activity of caspases during the onset of apoptosis in vitro. We describe how these probes can be used to biochemically profile caspase activity in vitro using fluorescent SDS-PAGE as well as their application to imaging protease activity in live animals and tissues.
Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC, Australia. laura.edgington-mitchell@monash.edu.
Laura E
LE
Edgington-Mitchell
Department of Pathology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA, USA.|Department of Microbiology and Immunology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA, USA.
Matthew
M
Bogyo
D000818:Animals; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism*; D003110:Colonic Neoplasms / Q000201:enzymology / Q000473:pathology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D051379:Mice; D057054:Molecular Imaging / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
D005456:Fluorescent Dyes; D020169:Caspases
Activity-based probes; Apoptosis; Caspases; Fluorescent SDS-PAGE; Imaging; Proteases