protein not stained by Coomassie Blue - (Oct/15/2012 )

Yes, I'm sure I have used the same bottle of Coomassie (and I have never used R-250 before, so there is no bottle R-250 stock on my lab table).
My colleague found out that cobalt ions might interfere with Coomassie staining, so maybe some cobalt eluted from the column with my protein.

The protein did stain well on nitrocellulose with Ponceau, though. At least we are sure enough now that the protein is indeed present.

Thanks for your tip on the silver staining, I will try it!

So what did you do exactly with this silver staining? Did you repeat all steps including fixing?

1. Fix in 50% ethanol / 10% acetic acid
2. Fix in 30% ethanol
3. Sensitize
4. Stain
5. Develop
6. Stop

(I used the Fermentas PageSilver kit for silver staining)

we used a modified (by us) method of merril, et al (science, 211, 1437-1438 (1981)).

whether we destained first or not, we start restaining from the sensitization step. the proteins are already fixed and the sds is already removed (ethanol step).

Ok, thanks a lot! I will let you know if it helps.