Hi,
I also encounterd the same kind of problem with my protein. I noticed
that the protein eluted from Ni-NTA with 150mM imidazole or 300mM didn't
behave in the same manner; ie on gel filtration experiments, the 300mM
eluate totally came in the excluded volume while the 150mM eluate came
in the expected elution volume for a monomeric protein (at least for a
few hours). Finally, the total protein was aggregated the day after.
Using DLS experiments (dynamic light scattering), it was shown that the
imidazole induced the aggregation of a monomeric sample.
No reversibility was observed whatever I added in the aggregated form.
Finally, I chose to elute my protein with a pH gradient with Na acetate
buffered from pH 6 to 4 but without any imidazole.
This new protein is really less sensitive to aggregation (but much more
to proteolysis !!! With the protein eluted from imidazole gradient, the
protein was stable on months but was completely aggregated!).
Good luck
Valerie
Artem Evdokimov a écrit :
Jacob,
You can try several things, including the stuff already mentioned by
others – EDTA, salt, etc. A very useful option to keep in mind is to
check that excess imidazole isn’t causing the problem. You can find
out by simply diluting the fractions down, or by changing the resin.
His-SELECT and Co-Talon are two resins that elute in reduced amounts
of imidazole – for example, HisSELECT is washed typically with 10 mM
and eluted anywhere from 10 to 200, usually around 50 mM. In several
cases that I personally worked on, switching from Ni-NTA to one of
these resins eliminated all the issues. Finally, if for some reason
Ni-NTA has won your heart and you’d rather not abandon it – you can
always put a desalting column in line with the IMAC, this way the
protein would come out and get instantaneously exchanged into the
buffer of your choice.
Happy purifying,
Artem
* From: * CCP4 bulletin board [mailto:[EMAIL PROTECTED] *On
Behalf Of *Jacob Wong
*Sent:* Thursday, February 14, 2008 7:26 PM
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* [ccp4bb] rescuing crashing-out protein eluted from Nickel
column
Dear all,
I just ran into this problem and would like to see if I could get some
helpful tips before my protein completely crashes out.
I have a protein as 6His fusion and it remained bound to the Ni resin
with 40 mM Imidazole wash (added to 1XPBS) but then was eluted off
with 200 mM (added to 1XPBS). The protein seemed to be highly
concentrated in the elution and began to get cloudy right away, with
more and more precipitation produced over a matter of minutes. I felt
so helpless, didn't know what to do, and then decided to add 5% of
glycerol into one of the fractions but that made it even more cloudy
(ohh no...).
While the protein is dying in the tube, do you have some quick remedy
for me? Thanks very much, -J.J.
