helicase fragment purification

Kyle Legate legatek at hotmail.com
Tue Apr 18 16:51:50 EST 2006


Bazeille Nicolas wrote:
>                              Hi all,
> 
> 
> Nowadays, I try to purify a his tag protein. I've already made experiments to
> estimate, using FPLC, the elution profil. After this elution, I need to
> precipitate my proteins to change the buffer (50 mM Tris-Cl pH9 500 mM NaCl,
> 500 mM Imidazole, 10% Glycerol => 50 mM tris-Cl pH 7.5 100 mM NaCl, 2 mM EDTA,
> 1 mM DTT) in order to load my proteins in heparin column. Yesterday, I've done a
> Ammonium sulfate precipitation after elution from NI2+ column, using 0.7 vol of
> a pre-cooled solution saturated by (NH4)2 SO4. After a stirring of two hours in
> ice, I could see floating protein precipitate. I've tried to rescue my proteins
> in a small tube and I've tested to solubilize its in my first buffer (50 mM
> Tris-Cl pH9 500 mM NaCl, 500 mM Imidazole, 10% Glycerol), but it's was
> impossible (denaturated). I really don't understand why this such situation
> have occured. I've read that it can resulted of a too high solution density. Is
> there a solution to avoid this kind of problem. I know that I can make a
> dialysis to eliminate Imidazole but I will need to make just after another one
> to eliminate AS. Because I'm not sure of the stability of my protein at +4°c
> during several nights. I believe in someone to help my to find a solution to
> precipitate my protein in the original buffer with a powder a AS.
> 
Forget the precipitation, just do dialysis. Several changes of dialysis 
buffer to lower the salt/imidazole/glycerol concentration, maybe 
periodically pH the whole setup to maintain pH 7.5 (remember, Tris pH is 
temperature sensitive so if you're doing everything in the coldroom make 
sure you pH your stocks when they're cold), and if your protein is happy 
without DTT  during the dialysis, omit it until just before you load the 
heparin column.


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