helicase fragment purification

Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Tue Apr 25 12:18:59 EST 2006


Bazeille Nicolas wrote:


> 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.

Ammonium sulphate precipitation needs to be done with carefully chosen
concentrations of the salt. You determine the concentration where your
protein is still soluble, and the concentration where it completely
precipitates (if you have an antibody against your protein, this is done
easily with a dot blot of precipitate and supernatant at various
AS-concentrations, e.g. 20, 25,...80% saturation). Add AS to the former
concentration, remove the junk by centrifugation and then add more AS to
the latter concentration. Spin again to collect your protein. 70%
satturation is already a quite high concentration, and may result in
solutions with higher density than that of your protein, as you
experienced. Excessively high AS concentrations also increase the risk
of protein denaturation.

However, in your situation I would probably concentrate the sample by
ultrafiltration and then use either ultrafiltration or gel filtration
for buffer exchange. Depending on the volume of your protein solution
ultrafiltration can be done either in centrifugal concentrators or in
pressure cells. Amicon used to make such devices, but was bought by
Millipore, IIRC.


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