If you really tried all sorts of buffers (did you go to extreme pH-values,
very high salt concentrations, tried various additives (beta-ME,
glycerol,...), different salts)
you can still
- try another expression system (insect cells, mammalian)
- see if any modifications might be useful
- try restricted proteolysis - well, you need a little bit of purified
protein for this, but it might be the most promising
- guesstimate a proper subclone from secondary structure prediction of
your
protein.
Tim
--
Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen
GPG Key ID = A46BEE1A
On Sun, 2 Mar 2008, Naren sharma wrote:
Hi all
sorry, for offtopic query...
I am trying to purify my protein by Ni-NTA affinity chromatography. After
sonication as i centrifuge bacterial lysate, soon after 10 min whole lysates
get precipitated during loading on the column and some time it remain
soluble too. if i get purified through the column without precipitation, it
gets precipitated during dialysis.
I have tried lot, by chnaging buffers, increasing salt or deacreasing salt
or no salt at are helpless.
I do purifiaction in cold room.
can any one suggest some solution?
Thanks in advance.
NSH
