solubility of protein - (Apr/14/2005 )

i wondered at which stage you can know the protein is insoluble?
after break the cells and centrifuge? if the protein stays in pellet, is that means not soluble? how do you test the solubility of expressed protein before large scale of purification.

Insoluble proteins are found as inclusion bodies and ypou can see them as black precipitate when you pellet your cells by centrifugation. To test for solubility of the expressed protein in E. coli run on the SDS-PAGE a lane for pellet and a lane for the subernatant if you see a big band in the pellet, the protein is mostly availble in inclusion bodies, if the protein is mostly appundant in the supernatant fraction it is a good indication that you have expressed your protein in a soluble format. Few suggestion I might add here, if you have your protein in inclusion bodies you can use Guanidine to solublize inclusion bodies or commercially availble kit such as the one Pierce has. In order to reduce the inclusion bodies formation, I would suggest transfering your cells after induction to a grow at 20C while expressing with gentle shaking (100 rpm).

Good Luck!