helicase fragment purification

Bazeille Nicolas nbazeill at etudiant.univ-lr.fr
Mon Apr 17 15:52:04 EST 2006

                             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.

Thanks a Lot


Trainee in LBPA Cachan (94230) France.

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