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Actin Bands smiling on western blot - (May/06/2011 )

Hi there, new to this forum but hope someone can help. i am running 12.5% next gels and have been goung for some time with no problems. The past couple of months i have been finding that my specific band of interest is fine, ponc S staining of the mebrane gives nice staining but the actin bands all have a big smile. i have tried running at 50-100V but still seem to be getting this problem. the gel comes ready prepared and had its own running buffer so i can't blame that either. Any ideas/suggestions?
many thanks
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How much sample volume did you load per lane?
It seems that you have loaded too much and some of your samples flow to the neighbouring well. :rolleyes:


It could be too much salt in your lysis or loading buffers.


I agree with kkmans that your wells are overloaded with protein.