SDS-PAGE with 500 ug protein - (Mar/28/2011 )

HI

I am trying to perform a 2D analysis with 500 ug of protein (12% PAGE). I know that the protein to SDS ratio has to be 1:1.4 Can I increase the SDS in the resolving and stacking buffers since I cant alter the amount at the sample stage? Please help. Krith

you shouldn't need to increase the sds in the buffers.

have you tried running with the normal formulation? if so, what happened that makes you think you need more sds?

I have tried running similar samples on 1D and found that they didnt move much. The bands remained at the top as blobs, right after the stacking gel front. I thought adding more SDS to the gel buffers would help me with the problem in 2D.

what is the expected molecular weight range of the proteins?

are you sure that 12% is not too restrictive?

have you tried a lower acrylamide concentration or a gradient?

are you sure that the protein is solubilized? did you boil in sample buffer?