Protein Purification-catastrophe while purifying? - (Sep/12/2013 )
I just added fresh bacterial culture to a previously pellet that was stored overnight at -20C and spun down the bottle to get a larger pellet. Question is whether the bacteria at -20C was already lysed and I lost my protein from the first pellet when I threw out the second supernatant? thanks for your advice.
Depends on cells and protein, but: Freezing is an effective lysis method. If the frozen pellet was thawed by the second suspension you probably washed out the protein.
You probably lost some protein having lysed the cells in your first pellet by freezing at -20C, but the loss shouldn't be too dramatic. I would still go ahead with the purification.