Help, protein ladder didn`t separate - (Jan/14/2014 )
I used a home-made 6% gel to separate out a protein of about 120kd but I did not get any separation of the ladder or anything else. I`m going to be checking the pH of all buffers, but as both stacking and resolving gels were solid, I don`t think the APS is a problem. Another person in the lab made gels the same day I did, and her`s resolved beautifully.
Further information, the pH of the stacking buffer was 7.0 rather than 6.8 so I will be making new buffer there. The resolving buffer was 8.86 rather than 8.8, so I`m still debating...
Those pH's aren't too far out, so I don't think that will be the problem. It could be that you forgot to add SDS?
was there any migration?
if not, check that power is getting through your apparatus (power supply may be faulty, cables may be faulty, electrodes may be broken or very dirty, could be insufficient electrode buffer to complete circuit, air in the sample wells).
you mention the gel buffers, what about the electrode buffer?
can you show us a picture of your gel?