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Now including Oncogene Research Products™ Data Sheet AP1029 Rev. 30-August-05 RFH Anti-Cytochrome c Mouse mAb (7H8.2C12) Cat. No. AP1029 Size: 50 µg Applications: Flow Cytometry (see comments) Immunoblotting (1 µg/ml) Immunocytochemistry (3 µg/ml) Immunoprecipitation (not recommended) Application Data: Lane 1: Detection of human cytochrome c by immunoblotting. Samples: Whole cell lysate (50 µg) from MCF7 cells. Primary antibodies: Anti-Cytochrome c Mouse mAb (7H8.2C12) (Cat. No. AP1029) (1 µg/ml). Detection: chemiluminescence. Lanes 2,3: Detection of human cytochrome c by immunoprecipitation followed by immunoblotting. Sample: Whole cell lysate (500 µg) from MCF7 cells. Antibody for immunoprecipitation: Anti-Cytochrome c Mouse mAb (7H8.2C12) (Cat. No. AP1029) (10 µg/ml) (lane 2) and Anti-Cytochrome c Mouse mAb (6H2.B4) (Cat. No. AP1030) (10 µg/ml) (lane 3). Immunoblotting conditions: same as lane 1. Detection of human cytochrome c by immunocytochemistry. Sample: HeLa cells fixed with 100% methanol. Primary antibody: Anti-Cytochrome c Mouse mAb (7H8.2C12) (Cat. No. AP1029) (3 µg/ml). Detection: fluorescence with DAPI counterstain. Description: Protein G purified mouse monoclonal antibody. Recognizes the ~15 kDa cytochrome c protein. Background: Cytochrome c (NP_061820) is a single copy nuclear gene that is translated on cytoplasmic ribosomes as apocytochrome c, translocates to the mitochondria and becomes combined with a heme group to form the holocytochrome c protein. Holocytochrome c is a soluble protein located in the intermembrane space of mitochondria, loosely attached to the inner membrane and is an essential component of the mitochondrial respiratory chain. Early studies showed that during the course of an apoptotic response there was a rapid loss of function of cytochrome c in the dying cell. This was later shown to be due specific release of cytochrome c from the mitochondria with subsequent accumulation in the cytoplasm. While the exact mechanism is still unknown, the release of cytochrome c has been shown to precede loss of membrane potential by several hours, occurring early before mitochondrial depolarization, caspase activation and DNA fragmentation. The efflux of cytochrome c is prevented by bcl-2 or bcl-xL resulting from the direct interaction of bcl-2 or bcl-x with the mitochondria. In separate investigations, cytoplasmic cytochrome c has been identified as Apaf-2, where it associated with Apaf-1 and caspase-9 to mediate the dATP-dependent activation of caspase-3. Using in vitro reconstitution systems, it has been shown that there is no species restriction on the cytochrome c used to generate the trimeric complex. Host: Mouse Immunogen: Equine cytochrome c Epitope: Within amino acids 93-104 Clone: 7H8.2C12 Isotype: IgG2b Known Species Reactivity: Horse, human, mouse, rat Positive Control: MCF7, P388D1, Hela, Jurkat, or NIH/3T3 cells Form: Liquid Formulation: In PBS. Please see label for lot-specific concentration. Preservative: ≤0.1% sodium azide Storage: REFRIGERATOR (+4°C). Avoid freeze/thaw. Toxicity: MSDS available upon request. Special Instructions: Following initial use, aliquot and freeze (-20°C) for long-term storage. Comments: Together with clone 6H2.B4 (Cat. No. AP1030) this antibody can be used to evaluate cytochrome c release from mitochondria during apoptotic cell death (see application references). Clone 6H2.B4 has been reported to recognize both mitochondrial and cytosolic cytochrome c while clone 7H8.2C12 detects mitochondrial cytochrome c but does not detect released cytochrome c. Detects only the denatured form of cytochrome c. Does not recognize the native form of cytochrome c. This antibody has also been reported to work for flow cytometry. Antibody should be titrated for optimal results in individual systems. References: Bossy-Wetzel, E., et al. 1998. EMBO J. 17, 37. Kluck, R.M., et al. 1997. Science 275, 1132. Yang, J., et al. 1997. Science 275, 1129. Krippner, A., et al. 1996. J. Biol. Chem. 271, 21629. Liu, X., et al. 1996. Cell 86, 147. Gonzales, D.H., et al. 1990. J. Bioenerg. Biomembr. 22, 753. Evans, M.J., et al. 1988. Proc. Natl. Acad. Sci. USA 85, 9625. Application References: Evaluation of Apoptosis Mohr, A., et al. 2004. Cell Death Differ. 11, 1153. Stahnke, K., et al. 2004. Apoptosis 9, 457. USA and Canada Tel (800) 628-8470 [email protected] Germany Tel 0800 100 3496 [email protected] United Kingdom and Ireland UK Freefone 0800 622935 Ireland Toll Free 1800 409445 customer.service @merckbiosciences.co.uk A Brand of EMD Biosciences, Inc., an Affiliate of Merck KGaA, Darmstadt, Germany www.calbiochem.com FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE. All Other Countries Contact Your Local Distributor www.calbiochem.com [email protected]