Native PAGE problem - Protein doesn't enter in resolving gel (Apr/02/2008 )

Hi everybody,

I have tried to run a native gel to see if my purified protein is a monomer or dimer. Theoretical pI of the protein is 6.1, so I've tried using usual PAGE buffers without SDS -- stacking gel pH 6.8, resolving pH 8.8, running buffer pH 8.3.

But after staining with Comassie I saw nothing, so the protein is remained between stacking and resolving gels.

The protein is 35 kDa, so I tried 10% gel.

What can I do, try higher pH buffers? How high? Or perhaps less concentrated gel?

Thanks in advance!

try the ornstein and davis formulation. you can find it by searching bioforum.