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2D SDS-PAGE - wave spots problem


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#1 qzz

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Posted 13 July 2009 - 03:35 AM

Hi all,
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:

Posted Image

Posted Image

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Don't you have any idea what the problem is? Thank you for your advice.

Experimental condition:

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

#2 neuron

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Posted 18 July 2009 - 10:20 PM

Hi,

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 ;)

Edited by amj2, 18 July 2009 - 10:21 PM.


#3 mdfenko

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Posted 20 July 2009 - 11:13 AM

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).

Edited by mdfenko, 20 July 2009 - 11:14 AM.

talent does what it can
genius does what it must
i do what i get paid to do

#4 fishdoc

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Posted 20 July 2009 - 11:36 AM

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.




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