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Could denatured proteins be stored at -20C (or -80C) until use (for SDS-PAGE)?


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#1 anemia

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Posted 23 April 2009 - 08:11 AM

One of my former colleagues performed like this:

Mix the sample with equal volume of 2X loading/sample buffer; boil the mixture (denaturation) at 95 C for 3 min; cool down the samples on ice for 5 min; store the samples at -20C (or -80C) until use.

I actually followed this protocol and had no problem to detect protein expression with Western blot. However, I am not sure if this is a good idea. Is there any literature support?

#2 mastermi

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Posted 23 April 2009 - 11:47 AM

I don't know any literature support, but I think there would be no problems.
I used protein samples for western blot which were stored that way for more than one year (-20C), and everything was fine!

#3 Carlton H

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Posted 23 April 2009 - 12:13 PM

The literature support woud likely be really old. I'd suggest looking into old papers describing the kinetics of protein folding. While there may not be a paper that says "proteins do not fold at sub-freezing temperatures" there will likely be some describing the kinetics with respect to temperature, and will show that low temperatures inhibit protein folding.

It's generally accepted that proteins under very cold conditions are more or less in stasis (most things are). If possible, I'd also store them in a medium which will freeze at -80*C, such that the protein molecules will be sterically hindered by the frozen solvent. Keep them as cold as possible just to be safe. ... That would be my advice.

Cheers,
-Carlton
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#4 anemia

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Posted 23 April 2009 - 11:23 PM

Your suggestions are very helpful. Thanks a lot!




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