urea-denatured protein precipitates after dialysis - (Apr/08/2010 )
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
-vblanche-
vblanche on Apr 8 2010, 01:57 PM said:
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
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
-Inmost sun-