Protein Refolding problem - (Feb/01/2008 )
I was purifying a protein which is expressed in the inclusion body. I purified it using Ni-NTA column with 8M urea in the buffer and washed with linear gradient of urea from 8M-0M. Then it was eluted with 20mM Tris, 400mM imidazole, 500mM NaCl, pH=8.0. Protein was soluble at this point. But when I dialysis this to change the buffer to 50mM Hepes, 50 mM NaCl, protein precipitated out. I also tried Tris buffer, same thing. I am just wondering what's the problem here. And since it was soluble at one point without any urea, is it safe to say at that point, the protein refolded well?
Any opinion and suggestions are appreciated.
-pontiffq-
QUOTE (pontiffq @ Feb 1 2008, 12:17 PM)
I was purifying a protein which is expressed in the inclusion body. I purified it using Ni-NTA column with 8M urea in the buffer and washed with linear gradient of urea from 8M-0M. Then it was eluted with 20mM Tris, 400mM imidazole, 500mM NaCl, pH=8.0. Protein was soluble at this point. But when I dialysis this to change the buffer to 50mM Hepes, 50 mM NaCl, protein precipitated out. I also tried Tris buffer, same thing. I am just wondering what's the problem here. And since it was soluble at one point without any urea, is it safe to say at that point, the protein refolded well?
Any opinion and suggestions are appreciated.
Any opinion and suggestions are appreciated.
to find the ideal refolding protocol is not easy; if there is no experience for a distinct protein, several protocols should be tried; I would try a protein refolding kit such as of Novochem;
independent on this, I would try various pH and a mild reduction condition (f.i. 1 mM DTT)
-The Bearer-
I would think your [NaCl] might be too low. Try a step-wise dialysis.
-swanny-