Sign up ×

We're producing some membrane proteins and they aren't amenable to freeze thaws even when we add glycerol. The proteins are solubilized in detergent above the cmc so they should be in micelle form in solution. Currently, they are being held at 4C. I'm curious what is the best way to store them for long term use?

share|improve this question
this sounds like a case by case issue,but i'd be interested to see what someone with experience would say. –  shigeta Apr 6 '13 at 3:35
Can't you just store the cells you use to produce the protein and thaw them when you need them to produce more? Like that you wouldn't need to store the protein itself for a longer time. –  suvidu Apr 8 '13 at 11:18
You could always just add sodium azide and keep them at +4, so they don't have to go through a freeze/thaw at all. –  MattDMo Apr 8 '13 at 15:32
@Suvidu, I would counter that if the purification protocol is a pain, it's a valuable investment to be able to scaleup effectively. –  bobthejoe Apr 9 '13 at 10:21
I've heard some MPs like lac permease can be scaled up, others are more delicate. do you have an assay, or even crude crystallization conditions? that would really help of course. –  shigeta May 10 '14 at 13:16

2 Answers 2

If i were to have a blind attempt at your question, it is never a good idea to freeze thaw any protein, not even cell lysates. Thats the whole point of glycerol use, which we use in our lab at 50% (v/v) to store bead bound proteins. Thats also why antibodies and restriction enzymes are stored in glycerol since freeze thawing antibodies dissolved in water often leads to their degradation, although you only tend to store stuff in glycerol, like restriction enzymes, if the concentration of the protein is very very high. One golden trick I use is aliquoting my samples and snap freeze them in liquid nitrogen and store in -80 freezer. I'm curious as to what happens with your protein, does it degrade after a freeze thaw cycle? (I'm aware that transmembrane proteins are notorious when it comes to storage/maintenance due to their awkward hydrophobic transmembrane region so its best to speak to an x-ray crystallography expert, if there is some one like that roaming around your research lab :), since they have face problems like this quite frequently and have excellent suggestions). Are your proteins GST tagged and bound to glutathione agarose beads? or you use HIS tags and nickel beads and elute the purified protein through imidazole? i suspect it is GST in which case its probably not a good idea to snap freeze although I have (somehow) had frozen glycerol beads at -20 and they were fine after use. I have had a friend talking about protein purification and the fact that protein isolation has to be done fresh so you might have to go down that root if your current method is not ideal. Also one last question, are you purifying you protein bacterially or they get secreted into the media? I suspect if its a membrane protein you are using the cell based system since if it was E. coli you were using (as suggested above) you could have just had your protein expressed through IPTG and just washed you cell pellet in STE buffer and spun it down hard and stored the pellet at -20, in which case would save you some time in the purification process. The use of Na azide is a good idea too but I only use it for primary antibody storage which is already dissolved in the block buffer such as 5%-BSA-TBST for WB and store the whole thing at 4 oC and Na azide is there to prevent BSA and the Ab going off.

The main problem many many people have, including my self is that the majority of times, over expressed protein is insoluble or just doesn't get expressed in certain systems so you are very lucky to have a working system.

This is all I know about protein storage. I hope this helps.

share|improve this answer

I work with membrane proteins and in my experience some proteins can be frozen and thawed without any problems, other proteins just don't like that. It's linked to the stability of the protein. If you really need to freeze the protein you may think about playing around with different detergents to see if you can improve the stability of the protein.

General rules: - freezing membrane proteins at -80°C without glycerol - avoiding protein freeze-thaw cycles, making aliquots before freezing - keeping them at 4°C only for short term use - after concentration always spinning the protein (10 min at 100k for example) to precipitate aggregated material. Aggregation causes aggregation, so you want to get rid of it.

share|improve this answer
Hello metionina. This is an informative answer. Can you, if possible, add reference to the protocol that you follow. –  WYSIWYG Jul 4 at 20:19
I couldn't find any published protocol, probably because every membrane protein behaves very differently. This is what I learnt working with membrane proteins and what people mostly do where I work. Then, again, every membrane protein is different and I suppose you could find what I said true, partially true or false for your protein. These are for me general things I would do when working with a new protein. –  metionina Jul 7 at 21:26

Your Answer


