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Cytochrome C Detection Western Blotting

Cytochrome C Detection Western Blotting - Western Blot Forum

Cytochrome C Detection Western Blotting - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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  #1  
Old 09-08-2008, 10:43 AM
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Cool Cytochrome C Detection Western Blotting



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
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Old 09-09-2008, 07:57 PM
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Default Re: Cytochrome C Detection Western Blotting

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.
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Old 12-02-2009, 02:31 PM
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Default Re: Cytochrome C Detection Western Blotting

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!!
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Old 12-02-2009, 05:26 PM
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Default 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.

Bryan
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Old 02-20-2011, 10:07 AM
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Default Re: Cytochrome C Detection Western Blotting

and according to you, which one is the best method for quantification / detection of cytosolic cytochrome c?
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Old 02-24-2011, 05:34 PM
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Default Re: Cytochrome C Detection Western Blotting

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