Many people prefer fresh competent cells for protein purification of their crystal structure. Is it really true that using fresh competent cells gives a better chance of getting protein crystals?
Hi Ajeet,
I don't think the age of (bacterial?) cells used for expression will affect crystallization. The impact is on the yield - older cells tend to lose their robust expression.
I had one project where we used the same -80 stock of transformed cells for expression for a few years, then one prep the yield dropped considerably, and after that the yield was miserable. Fresh transformation fixed the problem, but protein from any prep would crystallize as well as the other.
Good luck! -Christina
Old competent cells usually give low protein yield. It has no direct influence on probability of crystallization. The chances of obtaining well-diffracting crystals depends on the protein purification strategy employed and not on expression.
I have also seen cultures from glycerol stocks stored in the -80 give lower yields than freshly transformed cells. I have not seen lower yields from fresh transformation using older competent cells. What are the mechanisms responsible for the lower productivity of cultures from glycerol stocks vs freshly transformed cells? Evidence or references please!
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