title: Analysis of caspase activation during apoptosis.
abstract: This unit describes three methods for the detection of caspase activation as cells undergo apoptosis. Simple and relatively quantitative enzymatic assays are provided using suitable substrates. Because the various low-molecular-weight substrates available for these assays are not selective, however, the assays do not accurately distinguish between various caspases. Immunoblotting is described for following the activation of specific caspases. When coupled with subcellular fractionation, this method can provide large amounts of temporal and spatial information about caspase activation. Finally, affinity labeling protocols are provided for detecting active caspases in whole-cell lysates or subcellular fractions.
authors.affiliation: Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: S H
authors.initials: SH
authors.lastname: Kaufmann
authors.forename: T J
authors.initials: TJ
authors.lastname: Kottke
authors.forename: L M
authors.initials: LM
authors.lastname: Martins
authors.forename: A J
authors.initials: AJ
authors.lastname: Henzing
authors.forename: W C
authors.initials: WC
authors.lastname: Earnshaw
mesh_terms: D000345:Affinity Labels; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D019921:Biotinylation; D020169:Caspases / Q000032:analysis / Q000502:physiology*; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000201:enzymology; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000032:analysis; D015151:Immunoblotting / Q000379:methods*; D007124:Immunoenzyme Techniques / Q000379:methods*; D009842:Oligopeptides / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D000345:Affinity Labels; D005456:Fluorescent Dyes; D009842:Oligopeptides; D020169:Caspases