PROTOCOL: Western Blot Detection of Cytoplasmic Cytochr

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A basic protocol and the required reagents, unless otherwise noted, are found in the Cytochrome c Apoptosis Detection Kit (catalog # KA0772).

MATERIALS

> PBS (not included in the kit)
> 1X Cytosol Extraction buffer, 1mM DTT and 1X Protease Cocktail (prepare immediately before use)
> Mitochondria Extraction buffer, 1mM DTT and 1X Protease Cocktail (prepare immediately before use)
> Cytochrome C Antibody (0.2mg/ml)
> Organelle Markers (not included in the kit)

METHODS

1. Induce apoptosis by desired method and include vehicle-treated cells/animal (negative control).
2.

For cultured cells:	For tissue (fresh is recommended):
a. Collect ~5 x 10⁷ cells by centrifugation at 200 x g for 5 minutes at 4°C. b. Wash cells with 10 ml of ice-cold 1x PBS. Centrifuge at 600 x g for 5 minutes at 4°C. Remove supernatant. c. Add 1 ml of 1X Cytosol Extraction Buffer Mix containing DTT and Protease Inhibitors to the cells. Resuspend the pellet by carefully pipetting up and down with a pipette. Incubate on ice for 15 minutes.	a. Isolate the tissue of interest using standard dissection procedures. b. Wash with 10 ml of ice cold 1X PBS and mince the tissue with a scalpel or razor blade. c. Resuspend each 10 mg of tissue in 1 ml of 1X Cytosol Extraction Buffer Mix containing DTT and Protease Inhibitors.

3. Homogenize cells in a pre-chilled Dounce tissue grinder (or pestle homogenizer). Perform the task with the grinder on ice. In general, 30-50 passes is suggested for cells and 20-30 passes for tissue. Efficient homogenization depends on the tissue and cell type.

Note: To check the efficiency of homogenization, pipette 2-3 μl 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 lack the shiny ring, proceed to step 4. Otherwise, perform 10-20 additional passes using the Dounce tissue grinder and check under a microscope again to confirm adequate homogenization. Excessive homogenization should also be avoided because it can damage the mitochondrial membrane and lead to the inappropriate release of mitochondrial components, comprising experimental results.

4. Transfer homogenate to a microcentrifuge tube, and centrifuge at 700 x g for 10 minutes at 4°C. The pellet contains the nuclei, cellular debris and intact cells, whereas the supernatant contains the cytosol and mitochondria.
5. Transfer the supernatant into a fresh microcentrifuge tube, and centrifuge at 700 x g for another 10 minutes at 4°C to remove any residual nuclei.
6. Collect supernatant into a fresh microcentrifuge tube labeled Cytosolic Fraction, and centrifuge at 10,000 x g for 30 minutes at 4°C. The resulting supernatant is the cytosolic fraction and the pellet is the mitochondrial fraction. Centrifuge and collect the supernatant again to remove residual mitochondria.

Note: To remove any residual cytosolic components, consider washing the mitochondria pellet in 1ml of 1X Cytosol Extraction Buffer Mix containing DTT and Protease Inhibitors and centrifuge at 10,000 x g for another 15 minutes at 4°C.

7. Resuspend the pellet in 0.1 ml of Mitochondrial Extraction Buffer Mix containing DTT and protease inhibitors, vortex for 10 seconds and label tube as Mitochondrial Fraction.

Note: Collected fractions can be stored at -80°C until use. Avoid freeze/thaw cycles.

8. Load 10 μg each of the cytosolic and mitochondrial fractions isolated from uninduced and induced cells on a 12% SDS-PAGE. Then proceed with a standard Western blot procedure and probe with the cytochrome c antibody (recommended working concentration is 1 μg/ml) and the proper organelle markers.

Note: The appropriate organelle controls should be used to confirm the purity and integrity of each fraction. Beta-actin (catalog # NB600-501) is a recommended cytoplasmic marker whereas VDAC1 (catalog # NBP2-38163) is a recommended mitochondrial marker. For more information, see our Antibodies for Organelle Markers Handbook at novusbio.com/om-handbook.

Learn more about Western Blotting

PROTOCOL: Western Blot Detection of Cytoplasmic Cytochrome c

MATERIALS

METHODS