I am having a problem running acetone precipitated protein on an SDS-PAGE gel. This is protein precipitated from bacterial culture supernatant. Using acetone, it appears to yield a visible protein pellet. However, I am having trouble getting this sample, after re-suspension in sample buffer (laemlli), getting any protein to show up on my SDS-PAGE gel. I am boiling the protein samples. My bradford assay indicates that there is roughly 2ug/ul in the sample. I am centrifuging after boiling to clear any aggregates or non suspended proteins.
When I stain the gel with CBB, it looks like a deep blue band on the bottom of the stacking gel, indicating that perhaps the protein never actually entered. My ladder runs fine, so I doubt that the gel itself is the problem.
Does anyone have experience with sample precipitation utilizing acetone? Most of my protocols say that I shouldn't dry the pellet too much, others indicate that it should be lyophilized before use. Seemling contradictory. Anywho, any insights would be appreciated.