title: Detection of caspase activity using antibody-based techniques.
abstract: A number of antibodies have been generated that recognize caspases from mammalian model organisms. These include antibodies that recognize specific caspase pro-forms and others that bind caspase cleavage fragments. These antibodies are excellent reagents for identifying which executioner caspases have been activated following application or induction of a specific apoptotic stimulus. This approach is more difficult to use with initiator caspases, however, because cleavage does not necessarily correlate with caspase activation. In this protocol, cultured cells are treated with a proapoptotic stimulus, and then protein lysates are prepared from the treated cells. The proteins are then separated by gel electrophoresis and transferred to a suitable membrane. The fragment-specific antibodies that recognize executioner caspases are used in a western analysis to determine the extent of activation and to aid in identifying which caspases have been activated.
authors.affiliation: Department of Life Sciences, New York Institute of Technology, Old Westbury, New York 11568;
authors.forename: Gavin P
authors.initials: GP
authors.lastname: McStay
authors.affiliation: Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105.
authors.forename: Douglas R
authors.initials: DR
authors.lastname: Green
mesh_terms: D015153:Blotting, Western; D020169:Caspases / Q000032:analysis* / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D019169:Jurkat Cells
chemical_list: D020169:Caspases