Download Anti-Cytochrome c Mouse mAb (7H8.2C12) Cat. No. AP1029

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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.
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