Cytochrome c in SDS-PAGE - (Sep/20/2008 )
Dear all,
As I has purified cytochrome c from the cells, then run SDS-PAGE to see the purity. There were several bands in the gel. However, some teammates told me it was usual because cytochrome c might form the complex such as dimers, trimers..even pentamers.
However, i didn't agree with them. Since all proteins were performed SDS and mercaptoethanol before loading into the gel..
CAn anyone tell me the explaination...
THanks alot
Multimers of protein can occur even in SDS-PAGE, if the samples are not properly reduced, perhaps DTT was too old or the concentration too low, boiling too short, etc. But normally there should not be so much of the multimeric forms present. If you reduce very long and properly, these forms should disappear. If not it is something else.
The antibody I used to use (from BD) to detect cyt c also used to pick up a band at approx 60kDa.
It did this even in cells null for cyt c.
I used it as a psuedo loading control to check that the same amount was in all my lanes.
What antibody are you using?
It did this even in cells null for cyt c.
I used it as a psuedo loading control to check that the same amount was in all my lanes.
What antibody are you using?
Yes, I also. Do you know the reason? Is it really related to the multicomplex of the cytochrome c>>??
It did this even in cells null for cyt c.
I used it as a psuedo loading control to check that the same amount was in all my lanes.
What antibody are you using?
Yes, I also. Do you know the reason? Is it really related to the multicomplex of the cytochrome c>>??
Considering as I said before I saw the band in cells null for cytochrome c (not just knockdown, knockout) I would assume that it has nothing to do with multicomplex of cytochrome c and is just a non-specific band that is picked up by the antibody. I did contact BD about it once and they had no further information to provide me with.
how did you purify cyt c from cells in first place? it's not easy....
I think clone 7H8 detects a band as low as 10KDa....but I've heard many others detecting more than 1 band for cyt c....I know someone on this forum who detected one band at around 70KDa and another around 15KDa.