Ugly smears in SDS-PAGE after MeOH precipitation - (Feb/25/2008 )

Hi to all, I have a problem with smears in methanol-precipitated samples in SDS-PAGE.

I make a methanol precipitation from medium, than I centrifuge and dry pellets. Than I solubilize it and boil with standard Laemli Sample Buffer for SDS-PAGE. When i load it into a 8-16% gel wells, i have a ugly smear from the bottom of the well until half part of the gel.. and in Western blot too coloured with red Ponceau..

Are there some precipitates? the sample appears a bit cloudy....

thanks for answer..

S.75

If it seems to be cloudy perhaps it would help to centrifuge the samples prior to loading them on the gel. Perhaps they are not fully denatured or the amount of protein is very high and you have to use more sample buffer for solubilization.

Thanks, i will try both..

s.75