We have neutral protease, which gets denatured in a week even after freezing.
The way the protein is frozen is as important as how it is stored. Protein solutions should be snap-frozen, not allowed to freeze gradually. The reason is that slow-freezing allows separation of the protein and dissolved salts from the ice. The ice ends up at the top and the protein and salts end up at the bottom. Use small aliquot volumes and freeze them by immersion of the tubes in dry-ice acetone bath, dry ice-ethanol bath, liquid nitrogen, or powdered dry ice.
Do not store proteins in phosphate buffer at -20oC.
Storage at -80oC or in liquid nitrogen is highly preferable to storage at -20oC.
Freezing is a process that denaturates proteins, obviously it depends on the stability of a particular protein, most of them they are either very stable or are able to refold when brought back to room temp. You can help it by using a cryoprotectant as Glycerol (20% to 40%) and store it at -80, that should increase the life of your enzyme.
Hi Jeeva,
I would say yes because it depends on the number of times of freeze-thaw cycles before you finally store it at -20degrees and also microbial contamination can also degrade your protein if it was prepared by you.(Did you handle it under sterile condition before storage).Finally it also depends on the concentration of your enzyme because if they are of low conc and stored at smaller volume,they are prone to denaturation than larger ones(hydrophobicity of plastic tubes).
I hope this will help.
The enzyme doesn't get denatured in frozen condition. It's activity is preserved in freezing conditions. However, when it is brought back to room temperature, it does display lesser activity than before freezing. As mentioned above, a cryoprotectant like glycerol remarkably preserves enzyme activity in the freezing state as well.
Hope this helps.
The way the protein is frozen is as important as how it is stored. Protein solutions should be snap-frozen, not allowed to freeze gradually. The reason is that slow-freezing allows separation of the protein and dissolved salts from the ice. The ice ends up at the top and the protein and salts end up at the bottom. Use small aliquot volumes and freeze them by immersion of the tubes in dry-ice acetone bath, dry ice-ethanol bath, liquid nitrogen, or powdered dry ice.
Do not store proteins in phosphate buffer at -20oC.
Storage at -80oC or in liquid nitrogen is highly preferable to storage at -20oC.
The denaturing of your enzyme after a week of storage at -20 oC is rather strange. Perhaps you might want to check with the supplier, the preferred storage condition. However, I do believe the freezing-thawing cycle can reduce the activity of the enzyme after a number of times. The freezing process itself alone should not affect the structural integrity of the enzyme.
It is not strange, definitely storing at that condition could affect the stability of the enzyme. Even stock organisms you don't store them at that condition. You can only do that if you already know the stability of the enzyme and how it impacts on its activity. However, is it a published practice or standard to store at that condition? If yes, are you working with the same enzyme?
Adam Shapiro's answer is perfect. Mind you even storage in buffer at -80 degree impacts the activity. Use it only when you have underscored the effect on your enzyme stability and activity. But based on my experience, better recommendation is freeze drying in liquid nitrogen and store at -80 degrees afterwards. Don't know, I have not tried in glycerol before. Otherwise freshly prepare your enzyme and use it immediately...that's the hard but best practice!
Alternatively, if you are working with bacteria (microorganisms), you can store your induced cell pellets at -20 or -80 degrees after 2-3 times buffer washing to remove excess medium (careful not to lyse the cells mechanically). Store not more than 4 weeks. Then when you need the enzyme you start from the frozen cell pellets. Mind you its not a blanket rule
Cheers!
Dear Jeeba,
I hope that attached paper can be useful for you.
Yours
Hector
I agree with Adam Shapiro's response, as I am a Principal Scientist from MilliporeSigma. Rapid freezing is a method that we use to stabilize the enzyme the best, in the same manner that Adam described. At our facility we of course have large equipment using Glycol baths to free the enzyme solution in at little as 20 minutes. The key is to have an enzyme at the optimal protein concentration that will also help stabilize it in the freezing and freezer storage process. Of course, once frozen if you do not have access to a -80 C freezer, you will at least need a Freezer capable of -20 C which does not go through defrost cycles. Also, another option is to add stabilizers to your enzyme such as Trehalose.
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