Ghost band in SDS-PAGE - Help me!!!! (Mar/20/2006 )
naive question :
did you boil just before to load,
or did you boil, freeze your sample, and load later?
did you boil just before to load,
or did you boil, freeze your sample, and load later?
Usally I jest boil before to load...
Boiling at very high temperature ie 100C creates aggregates.
Heat at lower temperature for less time (ie less than 5 min)
Heat at lower temperature for less time (ie less than 5 min)
I tried also to don't boil at all and nothing changed that band was always there!
Any other suggestions? please!!!!!!!!!!!!!!!!!!!!!!!!!!!
could the DTT solution be too old?
Is your protein purification protocol quantitative or are there some other proteins known to be copurified??? Are you sure that 30kDa is a dimer or a different protein?
From my group I know the problem that often the purification reveals some other proteins, sometimes wich double or triple mass and than we thought about aggregation until we know the real identity of the band (via MS)!
Oli
We have just bought DTT and also when I prepare the new sample buffer solution with DTT 3.1mg/ml that band is always there!
I'm quite sure that I haven't any other proteins in my sample!!!!!
Thanks guys for your suggestion!!!!!!!!!!!!!!!!!!!!!!
Some problems with the cloning strategy, maybe double insert or something like that?
umm....no....
I didn't express this protein, I purifed it from biological fluid!