2D SDS-PAGE - wave spots problem - problem with pattern distorsion - "wave spots" (Jul/13/2009 )
I have a problem with my 2D SDS-PAGE. There are pattern distorsion in my gels - it seems that spots (less than about 30 kDa) make "waves" (but not all gels have this problem in spite of the same experimental conditions).
There are some pictures:
Don't you have any idea what the problem is? Thank you for your advice.
sample - depleted plasma (7 most abundat proteins except fibrinogen), desalted, concentrated
strips equilibration overnight (with sample), after first dimension 20 + 20 min equil. with DTT and iodoacetamide
second dimension (gradient gels 5-15%), 30 mA/gel, about 1 hour runnig
Coomassie blue staining
I would say may be your IEF is not proper, have you tried increasing focusing time? And what apparatus are you using, BioRad or Amersham? Anyways it doesn't matter, but you must have got a booklet along with the apparatus.In that booklet all the possible patterns of distorted gels are given, you can match your gel and figure out your problem. Hope it Helps
i can't see your picture here at work (firewall) but if you are seeing what i think you are seeing then it can be caused by excess electrode solution on your buffer strip (cathode, i think).
How old are your gels? I used expired gels once, and they ran horribly. Everything else was fine, just changed the gels out and the images were much better.