SPECIFICITY

A cytochrome is an electron-transporting protein that contains a heme prosthetic group (reviewed in 1). Cytochromes have been known to be essential components of the mitochondrial respiratory chain since 1925. The iron atom of the heme group in cytochromes alternates between a reduced ferrous (+2) state and an oxidized ferric (+3) state during electron transport in oxidative phosphorylation. Cytochromes are classified into four groups (a, b, c, and d) according to spectrochemical characteristics, and there are five cytochromes between Coenzyme QH2 and O2 in the electron transport chain. Cytochrome c is a water-soluble peripheral membrane protein of the mitochondria and transports electrons from the Coenzyme QH2 - Cytochrome c reductase complex to the Cytochrome c oxidase complex. Recently it has been suggested that Cytochrome c may be required for cells to undergo apoptosis.(2) Mitochrondria may be involved in apoptosis by releasing Cytochrome c to the cytosol where it functions in the activation of Caspase 3 (CPP32), an ICE family molecule that has been shown to trigger apoptosis when activated. Cytochrome c migrates at a reduced molecular weight of 15 kD.(2)

Clone 6H2.B4 recognizes the native form of rat, mouse, and human Cytochrome c.(3) It does not recognize the denatured form of Cytochrome c. 6H2.B4 binds to a region around reside 62 of rat Cytochrome c, based on competitive ELISA results.(3) Rat Cytochrome was used as immunogen.(3) Applications for clone 6H2.B4 include immunoprecipitation (1-2 µg/one million cells).(2) Suggested positive controls for detecting Cytochrome c include P388D1 mouse lymphoma cells (ATCC CCL 46), HeLa human carcinoma cells (ATCC CCL2), Jurkat T leukemia cells (ATCC TIB 152), and 3T3 NIH/3T3 mouse fibroblast cells (ATCC CRL 1658). The antibody should be stable for at least one year when stored at 4°C.

Clone 7H8.2C12 recognizes the denatured form of pigeon,(3) horse,(3) rat, mouse, and human Cytochrome c. It does not recognize the native form of Cytochrome c. Clone 7H8.2C12 recognizes an epitope within amino acids 93-104 (inclusive) of pigeon Cytochrome c, based on competitive ELISA results.(3) Applications include western blot analysis (1-2 µg/ml). Suggested positive controls for detecting Cytochrome c include P388D1 mouse lymphoma cells (ATCC CCL 46), HeLa human carcinoma cells (ATCC CCL2), Jurkat T leukemia cells (ATCC TIB 152), and 3T3 NIH/3T3 mouse fibroblast cells (ATCC CRL 1658). The antibody should be stable for at least one year when stored at 4°C.

REFERENCES
1. Boyer, P.D., B. Chance, L. Ernster, P. Mitchell, E. Racker, and E.C. Slater. 1977. Oxidative phosphorylation and photophosphorylation. Ann. Rev. Biochem. 46:955-1026.
2. Liu, X., C. N. Kim, J. Yang, R. Jemmerson, and X. Wang. 1996. Induction of apoptotic program in cell-free extracts: requirement for dATP and Cytochrome c. Cell 86:147-157.
3. Jemmerson, R., J.G. Johnson, E. Burrell, P.S. Taylor, and M.K. Jenkins. 1991. A monoclonal antibody specific for a Cytochrome c T cell stimulatory peptide inhibits T cell response and affects the way the peptide asssociates with antigen-presenting cells. Eur. J. Immunol. 21:143-151.
4. Goshorn, S.C., E. Retzel, and R. Jemmerson. 1991. Common structural features among monoclonal antibodies binding to the same antigenic region of Cytochrome c. J. Biol. Chem. 266:2134-2142.

PharMingen also supplies a number of reagents for apoptosis research, including:

• APO-BRDU, APO-DIRECT, and Annexin V-FITC for detection of apoptotic cells by flow cytometry
• Antibodies to Bcl-2 family proteins
• Antibodies to Fas and Fas Ligand
• Antibodies to ICE family proteins
• Antibodies to tumor suppressor and signal transduction proteins
• Antibodies to transcription factors
• RiboQuant RNase Protection Assay System for mRNA analysis, featuring human apoptosis probe sets

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