Western blot analysis of Jurkat cell extracts untreated or treated with cytochrome c in vitro, showing full length and/or cleaved caspase-3 (upper) and cleaved caspase-3 Asp175 (lower), using Caspase-3 Antibody #9662 and Cleaved Caspase-3 (Asp175) Antibody #9661.
Boil for 3 minutes prior to use. Load 10 ul of untreated and cytochrome c treated Caspase-3 Control Cell Extracts per lane.
Supplied in SDS Sample Buffer: 62.5 mM Tris-HCl (pH 6.8 at 25°C), 2% w/v SDS, 10% glycerol,50 mM DTT, 0.01% w/v bromophenol blue or phenolred.Store at –20°C, or at –80°C for long-term storage.
Caspase-3 Control Cell Extracts (Jurkat Untreated): Untreated Jurkat cells are lysed in Chaps cell extract buffer and a cytoplasmic fraction is generated to serve as a negative control for caspase cleavage. Supplied in SDS sample buffer.
Caspase-3 Control Cell Extracts (Jurkat +Cytochrome c): Untreated Jurkat cells are lysed in Chaps cell extract buffer and a cytoplasmic fraction is generated. Extracts are treated with cytochrome c in vitro to generate a positive control for caspase cleavage. Supplied in SDS sample buffer.
Caspase-3 (CPP-32, Apoptain, Yama, SCA-1) is a critical executioner of apoptosis, as it is either partially or totally responsible for the proteolytic cleavage of many key proteins, such as the nuclear enzyme poly (ADP-ribose) polymerase (PARP) (1). Activation of caspase-3 requires proteolytic processing of its inactive zymogen into activated p17 and p12 fragments. Cleavage of caspase-3 requires the aspartic acid residue at the P1 position (2).