SDS PAGE problem - (Jan/18/2007 )

Hi all,

I recently run tissue lysate on 10% SDS PAGE. Apparently, After staining, the protein sample lane appeared to be streaky and broaden( across the entire vertical lane). I only load 10 ug, desalt and spin the sample before running. The running voltage was at 150V constant. Does anybody have any ideas or suggestions? Any help is appreciated. My email address djohan@agenica.com

Thanks,
Djohan

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any chance of a photo of the offending gel?

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I agree that looking at it would help understand better what happened. One first thoght is to check whether themachine is getting very warm while running at high voltages. I do 10%gels all the time, and I was told never to go higher than 120V, I go up to 100V, because there could be warming up. So try first either to run in a cold room or to lower the Volts. what you describe of course looks like overloading to me, which you say you don't do, so try this at the moment.Hope it helps

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Thanks for your reply. I have also attached the gel image for your reference. The protein std marker is on the far left hand side. The std lane looks fine. The next 4 lanes are 2 tissues lysate loaded in duplicate. They appeared to be broaden and streaky. The dye front is also wavy. I will try to lower the voltage on my next round, but I have a feeling it is not the problem. Anyway, many thanks.

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I would say you load too much detergent. what was the detergent, the concentration, and the volume loaded?
Maybe you could also improve the migration by using fresh SDS, but I would say the problem is the detergent.

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I would check all detergents you use, especially these in sample buffer. Remember, that also salt concentrations should't be to high.
Compare your gels with my results attachet as photo to one of the previous topic on this forum (SDS_PAGE distorted lanes).
Good luck,
Grzeniu

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