Has anyone any experience with the detection of cytochrome c in the cytosol? When doing western blot, I can see the band of cytochrome c very clear on my blot (~17kDa), but there is a lot of aspecific binding too. I was wondering, as cytochrome is part of a bigger complex, the apoptosome, if it is possible that I'm also detecting higher complexes where cytochrome c is included? I see an extra band between 160 and 210 kDa and lower bands between 50-60kDa.
Are these complexes conserved or broken down during cell lysis and protein collection?
Many thanks for your help
Over the past 18 years we have carried out many immunoblots to detect cytochrome c release from mitochondria during apoptosis. We do often, but not always detect higher MW bands as you discribed. The fact that these bands persist on SDS-PAGE suggests a possible covalent modification. as ubiquitination is the most common covalent modification, it prompted us to immunopricipitate cytochrome chrome c fooled by immunoblotting forubiquitin
I sent the last answer before I was finished. Here is the full answer.
We have carried out many immunoblots to detect cytochrome c release from mitochondria during apoptosis, over the past 18 years. We do often, but not always detect higher MW bands as you described. The fact that these bands persist on SDS-PAGE suggests a possible covalent modification of cytochrome c. As ubiquitination is the most common covalent modification of proteins, it prompted us to immunopricipitate cytochrome c followed by immunoblotting for ubiquitin. Indeed many of the higher MW bands corresponded with poly-ubiquitinated forms of cytochrome c. So I think what you have observed on your blots are poly ubiquitinated cytochrome c.
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