western blotting for cytochrome c - (Apr/05/2007 )

Has anyone here tried doing this? Im using an antibody from BD pharmingen and all i am getting is 2 high molecular weight non specific bands and nothing at the correct size of 15 kDa. I am assaying fractionated cells and there should be cytochrome c here. Would anyone have a protocol I could compare to? I am using a 15 % tris glycine gel, transferring at 0.2 amp X 30 min (semi dry). I did a coomassie on the gel after and it looked like all the low Mr proteins had left the gel - do you think they could have gone through the membrane that quickly? I did a quick stain with ponceaus after transferring and I didnt see any low Mr bands but I only loaded 10 ug of protein to start with so even the high Mr proteins looked very weak (although they had not transferred completely). For blotting I blocked with 5% skim milk in TBS-T, and applied primary o/n at 4 oC (1:1000) in TBS-T, seconday for 1 h RT in TBS-T and developed. Any suggestions welcome,
thanks

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what kind of blotting membrane do you use? with NC, a blotting through may be possible...

load more than 10 µg; in addition, cytochrom c in purified form is cheap and should be run as a control for blotting and quality of Ab (I could be possible that your Ab has low sensitivity)

if you have no pure cytochrom c, ask biochemists in your near who routinely use gel filtration; they may use cc to calibrate

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Hi,

I'm also having this problem. Have you found a solution to it?

Thanks

Cheers,
Jing

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We also use the BD antibody, to pick up the low kDa bands with this antibody you need at least 30ug of protein or you won't be able to detect them properly. 10ug is right on the edge of the antibody sensitivity!

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yes, my friends also use 1:250 for Cytochrome C for western blot.

right now I'm doing ImmunoFluorescent with cytochrome C 7H8, because I didn't have Digitonin to only get cytoplasm lysate, as I don't want mitochondria and nuclear lysate. I am using 1:100 dilution. hope it works.

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