Monoclonal Antibody to Cytochrome C (Clone 6H2.B4)

Background
In addition to its well-known role in electron-transport, cytochrome c (CYT) has been identified as a mediator of apoptosis or programmed cell death (1). Cytochrome c translocates from mitochondria into the cytoplasm in the early phases of apoptosis and plays a role in initiation of the apoptotic proteolytic cascade bay activating caspase-3 (2). Cytochrome c has been shown to bind Apaf-1. Apaf-1 has also been shown to activate capase-3 (3).

Antigen
Rat cytochrome c was used as immunogen to generate 6H2.B4 hybridoma clone (Goshorn et al, 1991). The antibody recognizes an epitope around residue 62 of rat Cytochrome c, based on copetitive ELISA results (Goshorn et al. 1991).

Application Notes
(1). Clone 6H2.B4 (IMG-102A) recognizes the native form of cytochrome c (Goshorn et al. 1991). Native cytochrome c has an approximate molecular weight of 15 kDa. (2). 6H2. B4 does not appear to recognize the denatured form of cytochrome c. Clone 7H8.2C12 (IMG-101A) is recommended for western blot. (3). 6H2.B4 (IMG-102A) can be used in combination with 7H8.2C12 (IMG-101A) for IP/Western blot analysis of cytochrome c. Please refer to the Figure. (3) Blocking: 6H2.B4 has been shown to inhibit caspase 3 activation when microinjected into cells. Users should determine concentration and conditions for their model systems.

Reference
1. Liu, X., Kim, CN, Yang, J., Jemmersen, R., and Wang, X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86: 147-157 (1996).
2. Fernandes-Alnemri, T., Litwack, G., and Alnemri, ES. CPP32, a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukin-1 beta-converting enzyme J. Biol. Chem. 269: 30761-30764 (1994).
3. Zou, H., Henzel, WJ, Liu, X., Lutschg, A., and Wang, X. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90: 405-413 (1997).