This antibody can be used at 2-4 μg /1 x107 cell equivalents for immunoprecipitation. For immunofluorescent staining, the suggested use of this reagent is ≤ 0.5 µg per 106 cells in 100 µl volume. It is recommended that the reagent be titrated for optimal performance for each application.
Application Notes:
Additional reported applications (for the relevant formats) include: intracellular flow cytometry5, immunofluorescence microscopy3,5, immunoprecipitation4, Western blotting4,5, and immunocytochemistry5.
Application References:
1. Goshorn, SC. et al., 1991. J. Biol. Chem. 266:2134. 2. Jemmerson, R., et al., 1991. Eur. J. Immunol. 21:143. 3. Chandra, D., et al. 2002. J. Biol. Chem. 277:50842. 4. Semenkova, L., et al., 2003. Eur. J. Biochem. 270:4388. 5. Shih, S-F., et al., 2001. J. Biol. Chem. 276:21870.
Cytochrome c was immunoprecipitated from Hela cell extract (1% NP-40) using 2-4 µg 6H2.B4 antibody/1 x107 cell equivalents. Immunoprecipitates were resolved by electrophoresis, transferred to nitrocellulose, and probed with the 7H8.2C12 anti-cytochrome c antibody. Proteins were visualized using a goat anti-mouse secondary conjugated to HRP and a chemiluminescence detection system. In addition to the specific 15 kD cytochrome c band immunoprecipitated by 6H2.B4, heavy and light immunoglobulin chains are recognized by the goat anti-mouse secondary antibody.
Description:
Cytochrome c is a 15 kD protein found in the mitochondrial intermembrane space with a heme-binding domain. Cytochrome c is a component of the electron transport chain; the heme group transfers electrons from cytochrome b-c1 complex to cytochrome oxidase complex. Cytochrome c initiates apoptosis by release to cytoplasm and binding Apaf-1 which activates procaspase 9. Cytochrome c interacts with the cytochrome b-c1 complex, cytochrome oxidase complex, heme, Apaf-1, and Caspase 9 proteins. The 6H2.B4 monoclonal antibody recognizes human, mouse, and rat cytochrome-c and has been shown to be useful for intracellular flow cytometric staining, Western blotting, immunoprecipitation, and immunofluorescence staining.
Other Names:
Cyt c
Structure:
Heme binding domain; 15 kD
Distribution:
Mitochondrial intermembrane space
Function:
Component of electron transport chain; heme group transfers electrons from cytochrome b-c1 complex to cytochrome oxidase complex. Initiates apoptosis by release to cytoplasm and binding Apaf-1 which activates procaspase 9
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