Why do I see three bands when probing for cleaved caspase-3?

It is possible to see three bands for the large subunit of cleaved caspase-3 due to cleavage at Asp175. However, the distinction between the three is the extent of processing at the amino termini of the proteins. (Reference: Fernandes-Alnemri T, Armstrong RC, Krebs J, et al. In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains. Proc Natl Acad Sci U S A. 1996;93(15):7464-7469. doi:10.1073/pnas.93.15.7464.)

Processing of caspase-3 into its fully active form is a two-step process:

• Step 1 involves cleavage at Asp175, which generates the 20 kDa large subunit and the 12 kDa small subunit.
• Step 2 occurs when the 20 kDa large subunit is further cleaved at Asp9, probably through auto-catalytic activity. The removal of the first nine amino acids of the pro-domain results in the 19 kDa subunit. 
• Further cleavage can also occur more slowly at Asp28 to generate the fully mature 17 kDa subunit. Accumulation of the 20 kDa and 19 kDa subunits is presumably due to inhibition of this processing. Moreover, the 20 kDa and 19 kDa subunits contain a portion of the pro-domain that can be bound by inhibitors of apoptosis proteins. (Reference: Paulsen M, Ussat S, Jakob M, et al. Interaction with XIAP prevents full caspase-3/-7 activation in proliferating human T lymphocytes. Eur J Immunol. 2008;38(7):1979-1987. doi:10.1002/eji.200838211)

Last updated: July 7, 2026