my protein does not freeze, help - (Aug/12/2005 )
i made recombinant protein and get it purified, finally it is in elution buffer containing 1M Nacl, Tris, 1M AMG( kind of sugar) and little EDTA, I added 10%glycerol, but the protein does not freeze. . Does anyone know the reason might be?
By the way, i am doing a histag protein too, if imidazole in it, can the protein be frozen? my boss said could, but i remembered that can not.
Thanks million for help
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Samples in 10% glycerol will not freeze at -20. Try putting it in the -80.
then how do you freeze your sample in -20 degree then?
I did freeze my protein sample in -20 and in glycerol.
I agree that 10% glycerol usually prevents freezing at -20. If you're used to freezing proteins in that condition, then you're glycerol concentration or temp are slightly different (or ours are).
That said, if you are used to freezing at that temperature, I could see the sugar lowering the freezing point - some sugars are used as cryoprotectants. Are you certain the sample isn't solid? Could be it just looks like liquid when you first take it out because the sugar caused it to freeze clear. It could also have lowered the freezing point (don't know the properties of the particular sugar).
Thanks for replying. Then how do you freeze your sample at -20 degree? or only freeze at -80? if freeze at -80, the protein will be only use once, right, then degraded. That will be a lost, so i prefer to use it more than once, that is why i choose to freeze at -20 degree. anyone can give advices on freezing at -20?
Is that true, I always thought that freezing at -80 will protect the protein much longer than at -20????
Anytime you freeze for multiple cycles you're going to experience problems with protein stability, whether its at -80 or -20. Ultimately it depends on the characteristics of the protein, but shifting them between solid and liquid phases too many times will result in loss of activity and/or aggregation. If you want to use a species multiple times, I'd suggest you aliquot your pure sample into smaller batches, then freeze them all at -80. You can then thaw one or two aliquots at a time for your experiments. You should find your results are more reproducible with this method.
Thanks a lot for replying. Do you add glycerol when you freeze at -80? have you frozen your sample in -20, if you do, how did you do that then.
Personally, no, because the primary species I work with is very stable. But for any unknown or new species I work with, it gets 5% glycerol in the storage buffer. I never freeze protein at -20, only -80.
Each protein is different. Ask yuourself why to you need to freez it first? There are other ways to preserve protein activities. Good luck!