I am recently working on a crystallization project and I have found the condition. But the sample i used have been stored at -80 by me stupidly..... and the protein have been freezed and thawed some times. Although it can still grow crystal, but it is never as good as the original sample ( the crystal now is scattered, small, instead of big). I tried to purify it again, but the newly purified protein cannot crystalize as the 1st batch. I want to ask can gel filtration separate the good protein from the bad one? Because I want to subject the 1st sample to gel filtration again, idk if that may help.....
Hi,
it might be good to at least try once to perform gel filtration with the sample you already have (or some part of it for a trial), and you might get rid of some sample heterogeneity. To check on that further you may preform a DLS (dynamic light scattering) before and after gel filtration step (to assess if it really helps). And then of course try to grow the crystals. Also take into account that the condition might differ still slightly from your previous one and you may need to optimise a bit.
Otherwise if that does not improve the situation, I would go for new expression batch and a freshly purified protein.
Hope that answers your question, good luck
Ilona
Apart from the options mentioned above, assuming you can grow many 'bad' crystals, you could try to harvest these and re-solubilize them as a 'purification by crystallization' approach to get rid of the bad. Then after re-solubilization set up crystallization experiments again and this may give you the better crystals hopefully.
Good luck, Tjaard
My suggestion would be to do a fresh purification, starting from freshly transformed colony. Next time after purification make small aliquots (to avoid repeated freeze and thaw).
Nothing wrong with -80oC storage, that's how I store my protein samples for crystallization, although multiple freeze/thaw cycles are frowned upon. Can you elaborate a bit more about your "newly purified protein"? What procedures did you use? Did it go through gel filtration? Anyway, gel filtration can certainly separate "good" proteins from "bad" ones, but only to some degree. In this case, gel filtration may separate aggregates from folded protein, but may not work well if there is minor degradation.
Another way to get large, good crystals is to try seeding.
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