Detection of Active Caspase 3 in Apoptosis

Caspase (Cysteine aspartic acid specificprotease) are a group of protease, which exist in cytoplasm. These proteasescan specifically cleave target proteins at C-terminal of aspartic acid residue,and their active site contains a cystein . Caspase play an important role inapoptosis, and 12 members of human caspase family were found till now.

Caspase 3 is an essential member of caspasefamily, it functions as the main terminal cleave enzyme in apoptosis. Caspase 3is generated from pro-caspase 3, which is a precursor protein, contains 277amino acid residues. It is found that the structure of pro-caspase 3 containsfour parts, the first one is 1-9 amino acid consisted propeptide, the secondpart is 10-28 amino acid consisted propeptide, the third one is a big subunitcalled p17, which consists of 29-157 amino acid, and the last one is a smallsubunit called p12, which contains animo acid of 176-277 (Fig.1). At thebeginning of apoptosis, pro-caspase 3 is activated by endogenous granzyme B andexogenous cytochrome C, it is cut into two subunits of p17 and p12 at two sites, Asp28~Ser29 and Asp175~Ser176. After that, the twosubunits will constitute active caspase 3, which is a hereotetramer, consistsof double p17 and double p12. The active caspase 3 could induce apoptosis aftercutting by its main substrate, PARP (poly (ADP-ribose) polymerase).

Dueto the importance of active caspase 3 in apoptosis, the detection of caspase 3and its activity are necessary. After analysis of the structure of caspase 3, Cloud-CloneCorp. had developed two subunits of the protein, the catalog number are RPA626Hu01and RPA626Hu02 ,respectively. The two recombinant proteins could be used while the researchersare studying the structure or activity of caspase 3.

Monoclonalantibody of MAA626Hu22and polyclonal antibodies of PAA626Hu01and PAA626Hu02are prepared afteranimal immunization. These antibodies could be used for semiquantitative andquantitative detection of active level of pro-caspase 3. There is an example ofexperiment, whose target is to determine the activationprogress and speed of caspase 3 by detection the concentration of pro-caspase3and p17/p12 (Fig. 2).

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