Detecting caspase activity in Drosophila larval imaginal discs

Methods Mol Biol. 2014:1133:109-17. doi: 10.1007/978-1-4939-0357-3_7.

Abstract

Caspases are a highly specialized class of cell death proteases. Since they are synthesized as inactive full-length zymogens, activation--at least of effector caspases and to some extent also of initiator caspases-requires a proteolytic cleavage event, generating a large and a small subunit, two of each forming the active caspase. The proteolytic cleavage event generates neo-epitopes at both the C-terminus of the large subunit and the N-terminus of the small subunit. The cleaved Caspase-3 (CC3) antibody was raised against the neo-epitope of the large subunit and thus detects only cleaved, but not full-length, Caspase-3. Although raised against human cleaved Caspase-3, the CC3 antibody cross-reacts in other species and detects cleaved caspases, most notably DrICE and Dcp-1, in Drosophila. This protocol describes the procedure for use of the CC3 antibody to detect caspase activity in larval imaginal discs in Drosophila.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Apoptosis / genetics*
  • Caspase 3 / genetics
  • Caspase 3 / isolation & purification*
  • Drosophila melanogaster / enzymology
  • Humans
  • Imaginal Discs / enzymology
  • Larva / enzymology
  • Molecular Biology / methods*

Substances

  • Caspase 3