Cytochrome C Detection Western Blotting

[darkwng]

Hey everyone

I have to detect Cytochrome c release before and after apoptosis as a marker of programmed cell death PCD.
Does anyone have or know of a specific protocol for mitochondria isolation and is RIPA isolation sufficient?

cheers

[wendyleye]

I had experienced a hard time to detect cytochrome c on western blot using the tris-glycine transfer buffer. Would you please keep me posted on how your experiment goes? Concerning mitochondria isolation, I know there're several mitochondria isolation and/or purification kits on the market for that purpose. I haven't used them myself, but I think QIAGEN's should be good.

[RLH]

I saw your old post and see that you were working with isolating and detecting cytochrome c from the mitochondria. I was hoping you would have some information on cytosolic cytochrome c as well.

Do you know what the significance is with detection of cytosolic cytochrome c?

I've been having an issue with my western blots- I see an increased expression in BAD, but the cytosolic cytochrome c is unchanged.

Have you worked on cytosolic cyt c? And do you know if increased BAD expression is always accompanied by increased cytosolic cyt c?

Thanks a million!!

[Warthaug]

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.

Bryan

[gehrs]

and according to you, which one is the best method for quantification / detection of cytosolic cytochrome c?

[nawi]

what type of transfer do you use, semy-dry or wet?