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Protein degradation

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Protein degradation

Postby ss_kalve » Wed Jan 30, 2008 3:46 am

I'm working with proteins.I stored my samples in -20C. But after some time i've seen that they r getting degrade.does any body know the reason?what should i do for long preservation of proteins?
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Re: Protein degradation

Postby blcr11 » Sat Feb 02, 2008 1:18 pm

It varies with the protein. Some are quite happy at 4C. Some will store OK at -20C, but usually you store proteins at -80C if you must freeze them. The problem with freezing at -20C is that the sample freezes slowly (minutes to hours) allowing ice crystals to mingle with the protein phase. When ice thaws, it expands and that can damamge protein. Also, at -20C there is still enough thermal energy for proteins to be mobile and spontaneously denature over time. At -80C the freeze-time is relatively fast, especially if you pre-freeze the samples in liquid nitrogen. The protein and water freeze in separate domains so that when the ice thaws, it has less chance to do damage to the protein. There is less of a chance for spontaneous denaturation at -80C compared to -20C. You can add glycerol to your sample (5-20% v/v is typical) to help with the freezing, but then you have to dialyze away the glycerol before using your protein, so you don't usually do that unless you know you have a protein that otherwise doesn't freeze well.

Should also add that if there is any protease contamination, any resulting degradation will likely be faster at -20C compared to -80C.
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Re: Protein degradation

Postby DrGonzo » Sun Mar 09, 2008 9:19 am

As far as I know, ice is not ice. There is ice I and ice II, what means that the crystal structure changes to the more packed structure of ice II around -20°C. Like most things in natural sience it is an equilibrium. That resulst a conformational shift of your protein, which can damage your protein. So if you need your protein constantly you have to store it at -20°C, then the impact of conformational change is not so massive by defosting and frezing. If you store it long time use the -80°C for the reasons named above.

A protease is not suppost to work either at -20 or -80°C, but if you can get rid of them, do it anyway! Cheers
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