| | Re: Cytochrome C Detection Western Blotting
The release of cytoc from mitochondria is considered to be the first irreversible step in apoptosis, so the detection of cytoC in the cytoplasm is usually taken as evidence that a cell is either apoptotic, or about to become apoptotic.
As for your issue with Bad, what you are seeing is not that unusual. Many of the pro-apoptotic proteins are in balance with anti-apoptotic proteins. Meaning that when you get a pro-apoptotic signal you may see changes in expression of pro-apoptotic proteins like bad, without seeing apoptosis itself. This occurs because there is still sufficient anti-apoptotic protein present to counteract the activity of the pro-apoptotic ones. In the case of bad, it can be inhibited by the activity of 14-3-3 proteins; so in order to get bad-mediated apoptosis you must also inactivate 14-3-3 proteins.