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?
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.
It could be too much salt in your lysis or loading buffers.
I agree with kkmans that your wells are overloaded with protein.