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urea-denatured protein precipitates after dialysis


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#1 vblanche

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Posted 08 April 2010 - 05:57 AM

Hello,

my his-tagged protein has a theoritical pI of 7.02.

So far, after expression in Origami, I've denatured the bacterial pellet in 6M urea/0.5M NaCl/20mM tris pH7.9.

I first tried a sequence of dialysis: 4M urea/PBS, 2M urea/PBS, then PBS. from 2M to PBS only, the protein precipitates.

Should I change the pH of my buffer?

any advice on stopping this precipitation?

Many thanks in advance.
vince

Edited by vblanche, 08 April 2010 - 05:59 AM.


#2 Inmost sun

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Posted 15 April 2010 - 12:38 PM

Hello,

my his-tagged protein has a theoritical pI of 7.02.

So far, after expression in Origami, I've denatured the bacterial pellet in 6M urea/0.5M NaCl/20mM tris pH7.9.

I first tried a sequence of dialysis: 4M urea/PBS, 2M urea/PBS, then PBS. from 2M to PBS only, the protein precipitates.

Should I change the pH of my buffer?

any advice on stopping this precipitation?

Many thanks in advance.
vince


I cannot offer a simple solution; I would try to run a renaturation protocol; if you have no idea, there are kits, f.i. from Novagen, to test nearly a hundred of different renaturizing protocols




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