Protein degradation

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ss_kalve
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Protein degradation

Post by ss_kalve » Tue Jan 20, 2009 12:44 pm

can anybody tell that after how much time protein at room temperature will degrade?

secondly, i've a doubt that after acetone precipitation i'm getting pellet which is not getting fully dissolved in resuspension buffer.what can be the reason and how can i dissolve the pellet of protein without any mechanical pressure......

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GreenDog
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Post by GreenDog » Tue Jan 20, 2009 2:31 pm

Could you be more specific?
Are you purifying proteins, how? Why would you want to leave them at room temp? During the day nothing will happen to them.
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ss_kalve
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Re: Protein degradation

Post by ss_kalve » Tue Jan 20, 2009 4:29 pm

Dear, thnx for reply.actually as i tld i've precipitated protein by acetone precipitation. In this precipitation method we get pellet which is protein and after centrifugation v have to collect the pellet and dissolve it into resuspension buffer.my problem is that my protein pellet is not getting fully dissolved. for that i kept it into waterbath for overnight at 25C. SO I WOULD LIKE TO KNW IS THIS METHOD IS CORRECT OR NOT? AND FINALLY I'M STILL NOT GETTING GOOD RESULT.

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GreenDog
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Post by GreenDog » Wed Jan 21, 2009 11:53 am

I wouldn't leave it on the bench o.n. however you can vortex and put in 37deg for half an hour or so. It actually depends on what you'll do next and on the bugger (for western we put for 5 min in 95 and it's ok, right?).
You could also try a different precipitation method.
"When In Danger Or In Doubt Run In Circles Scream And Shout"
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MrMistery
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Post by MrMistery » Thu Jan 22, 2009 2:34 am

Protein degradation also depends on the particular protein you're working with. Some proteins seem to be more sensible than others, though I don't know reasons (I mean, I can imagine some, but I never actually looked it up).
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canalon
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Post by canalon » Sat Jan 24, 2009 3:44 pm

Yep it is impossible to give you an idea of the stbility of a protein in general. Some buggers are near impossible to denature, like RNAses that easily survive autoclaving, while other start denaturing when you are extracting them.
And it depends of what you want: if you care a bout the activity, you have to be really careful, if you just want run in denturing gel stability is of little importance. As a rule I would rather put my tube in the fridge attached to some device that can agitate the tube and see if that works better.
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