Hi, I'm purifying Fab proteins but I have no time to finish it today so that I would like to continue the next day (I know i shouldn't but have no choice). Right now I'm centrifuging cell lysate so my question is: is it better to leave to clear lysate overnight in 4 degrees or to flash freeze in liquid nitrogen and store in -80?
We routinely store intact E.coli (BL21DE3 strain) in -80C before purification. In fact, freeze thawing would improve bacterial lysis. If you have already lysed the bacteria, you may want to add glycerol (10%) to bacterial cell lysate to protect from the denaturation/inactivation of your protein during storage at -80C.
We routinely store intact E.coli (BL21DE3 strain) in -80C before purification. In fact, freeze thawing would improve bacterial lysis. If you have already lysed the bacteria, you may want to add glycerol (10%) to bacterial cell lysate to protect from the denaturation/inactivation of your protein during storage at -80C.
It essentially depends on how sensitive your protein is. For sensitive proteins freeze thawing of the lysate is not recommended. In fact if you plan to subject your proteins to affinity chromatography keeping the lysate (after pelleting the bacterial debris) overnight in ice (preferably in the cold room) may be more sensible than freezing your protein at - 80 degree. As a thumb rule avoid freeze thawing of proteins as much as possible during the purification process especially if you plan to use them for activity assays as they may denature the protein of your interest. Also make sure your lysate has enough protease inhibitors. Also remember freezing bacteria and bacterial lysates (after you have lysed the bacteria) are entirely different ball games in protein purification.
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