Cytochrome c Apoptosis Assay Kit

Antigen: Cytochrome C, Somatic (CYCS)

Synonyms: cycs, CYCS, CYC, HCS, THC4, cyc, hcs, cyct, MGC75709, MGC89066, DKFZp468F1016, MGC128356, CYCSA, MGC93634, Cyt-c1, CytC-1, CytC1, Cytc, Cytc-d, DC3, bln, bs30f07.y1, cyt c-d, cyt-c-d, cyt.c, cytochrome c, ms(2)04445, pDMc02, DmelCG13263, CG13263, Cyt-c2, CytC-2, Cytc-p, DC4, DMc01, cyt c-p, cyt-c-p, l(2)SH1590, pDMc01, DmelCG17903, CG17903

Application: Biochemical Assay (BCA)

Catalog no.: ABIN411654

Additional Information

Alternative name: Cytochrome C

Description: Cytochrome c plays an important role in apoptosis. The protein is located in the space between the inner and outer mitochondrial membranes. An apoptotic stimulus triggers the release of cytochrome c from the mitochondria into cytosol where it binds to Apaf-1. The cytochrome c/Apaf-1 complex activates caspase-9, which then activates caspase-3 and other downstream caspases. BioVision’s Cytochrome c Releasing Apoptosis Assay Kit provides an effective means for detecting cytochrome c translocation from mitochondria into cytosol during apoptosis. The kit provides unique formulations of reagents to isolate a highly enriched mitochondria fraction from cytosol. The procedure is so simple and easy to perform, no ultracentrifugation is required and no toxic chemicals are involved. Cytochrome c releasing from mitochondria into cytosol is then determined by Western blotting using the cytochrome c antibody provided in the kit.

Synonyms: cycs, CYCS, CYC, HCS, THC4, cyc, hcs, cyct, MGC75709, MGC89066, DKFZp468F1016, MGC128356, CYCSA, MGC93634, Cyt-c1, CytC-1, CytC1, Cytc, Cytc-d, DC3, bln, bs30f07.y1, cyt c-d, cyt-c-d, cyt.c, cytochrome c, ms(2)04445, pDMc02, DmelCG13263, CG13263, Cyt-c2, CytC-2, Cytc-p, DC4, DMc01, cyt c-p, cyt-c-p, l(2)SH1590, pDMc01, DmelCG17903, CG17903

Application Details

Protocol: 1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction. 2. Collect cells (5 x 10 7 ) by centrifugation at 600 x g for 5 minutes at 4 o C. 3. Wash cells with 10 ml of ice-cold PBS. Centrifuge at 600 x g for 5 minutes at 4 o C. Remove supernatant. 4. Resuspend cells with 1.0 ml of 1X Cytosol Extraction Buffer Mix containing DTT and Protease Inhibitors (as prepared in Section A). Incubate on ice for 10 minutes. 5. Homogenize cells in an ice-cold Dounce tissue grinder. Perform the task with the grinder on ice. We recommend 30-50 passes with the grinder, however, efficient homogenization may depend on the cell type. Note: To check the efficiency of homogenization, pipette 2-3 μ l of the homogenized suspension onto a coverslip and observe under a microscope. A shiny ring around the nuclei indicates that cells are still intact. If 70-80% of the nuclei do not have the shiny ring, proceed to step 7. Otherwise, perform 10-20 additional passes using the Dounce tissue grinder. Excessive homogenization should also be avoided, as it can cause damage to the mitochondrial membrane which triggers release of mitochondrial components. 6. Transfer homogenate to a 1.5-ml microcentrifuge tube, and centrifuge at 700 x g for 10 minutes at 4 o C. 7. Collect supernatant into a fresh 1.5-ml tube, and centrifuge at 10,000 x g for 30 minutes at 4 o C. Collect supernatant as Cytosolic Fraction. 8. Resuspend the pellet in 0.1-ml Mitochondrial Extraction Buffer Mix containing DTT and protease inhibitors (as prepared in section A), vertex for 10 seconds and save as Mitochondrial Fraction. 9. Load 10 μ g each of the cytosolic and mitochondrial fractions isolated from uninduced and induced cells on a 12% SDS-PAGE. Then proceed with standard Western blot procedure and probe with cytochrome c antibody (1 μ g/ml is recommended).

Restrictions: For Research Use only

Publications

Publications: Suzuki, Akahane, Nakamura et al.: "Palmitate induces apoptosis in Schwann cells via both ceramide-dependent and independent pathways." in: Neuroscience, 2010 (PubMed).

El Hakim, Gamal-Eldeen, Shahein et al.: "Purification and characterization of a cytotoxic neurotoxin-like protein from Naja haje haje venom that induces mitochondrial apoptosis pathway." in: Archives of toxicology, 2011 (PubMed).

Saito, Nakagami, Azuma et al.: "Critical Roles of Cold Shock Domain Protein A as an Endogenous Angiogenesis Inhibitor in Skeletal Muscle." in: Antioxidants & redox signaling, 2011 (PubMed).