Hi Juliana,
Since you can purify some protein with IMAC, I suggest that you do a
buffer screening with Thermofluor before running the desalting column
(or gel filtration column) and use the buffer condition(s) that
stabilise your protein for the next purification step and possibly
freezing it in that buffer.
Good luck,
Vangelis
--
Vangelis Christodoulou
Analist C
Anastassis (Tassos) Perrakis lab
The Netherlands Cancer Institute
Division of Molecular Carcinogenesis - H2
Plesmanlaan 121, 1066 CX, Amsterdam
The Netherlands
tel: +31 (0)20 512 1951
fax: +31 (0)20 512 1954
Website: xtal.nki.nl/Tassos_group/
On Feb 15, 2008, at 17:47, Juliana Barbosa Coitinho wrote:
Dear all,
I am with the same problem of Jacob.
My protein is precipitating, especially when I 'freeze it'. I am
using a His Trap HP column coupled with a desalting column. Already
tried to elute with gradient of imidazole (that allowed an elution
with less amount of imidazole), but even then, the protein still
crashes-out.
I am trying to use the protein immediately after purified it, but
this is not always possible and I am losing much protein with that.
I will try to use the tips that have already been said.
But if someone can help me more, I will thank!!!
Thanks a lot!
Juliana
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