Protein samples still in liquid form at -20C, Storage condition for proteins

[sotellme]

Dear folks,

Please help me out. I store my purified protein samples at -20C and they are still at the liquid form (a few are frozen) at this temperature! I used Trizol kit to purify them and dissolved them in a solution of 9M urea, 4% CHAPS and 30mM Tris/HCl pH 8.5 buffer. They had very high concentrations to start with, but with time the concentrations are less than before. Can their liquid form degrade them? How can i store my protein samples better? At -70C? I have asked some people and they store theirs at -70C.

Thank you all for your kind help!

 

[DocToxyn]

It's fairly easy to achieve an effect called "supercooling" when you have very clean solutions with a fair amount of dissolved solutes, such as those you describe You can essentially cool them well below their freezing point since there is not starter crystals or dust or other contaminants in them to give the ice crystals a nucleus to form around. Sometimes all it will take is a quick shock, like flicking the tubes, and they will freeze. You could always snap freeze them in crushed dry ice or dry ice with ethanol and then put them in the -20 or just place them in the -70-80C freezer, they won't supercool that low.

 

[sotellme]

Do you have any ideas why my proteins have less concentrations now than before? Can this liquid form degrade them? Would it be harmful if they are still in this form or should they be better in the frozen form and stored at -70C?

 

[Moonbear]

We always stored ours at -70 C or -80 C just to be on the safe side. Some proteins are stable at -20 C, others will begin to degrade and need to be stored at -80 C. You also need to check that your protein isn't "sticky" and getting lost by sticking to the sides of your tubes during storage. Repeated thawing and refreezing can also degrade protein in your samples, so it is best to store multiple aliquots of a sample rather than thawing, taking out an aliquot to test, and putting the remainder back in the freezer.

Looking at the types of questions you've been asking for some time now, I'd really recommend you find another lab to work in or at least to learn the techniques in. You're making mistakes and still asking questions about things that should have been explained by your mentor/advisor in the first week to month in a lab. It's clear you're not getting the supervision you should have in your lab. On the other hand, these are the types of questions to ask before you begin. As you're about to put your samples into storage, that's when you should double check that the storage temperature will be correct, not after you take them out and find your samples have all degraded. As much as we try to be helpful here, we can't substitute for working with a knowledgeable mentor or planning ahead to make sure you have learned a technique before forging ahead with an entire experiment that depends upon it.

 

[sotellme]

Moonbear,

The reason why i stored my protein samples at -20C is because i followed this kit which said that the protein samples should be stored at -20C.

http://www.mrcgene.com/tri.htm

Thanks for your awesome tip. I will put them at -70C.