Did anyone suggest adding any known ligand? If your protein happens to
bind some compound/peptide/DNA/RNA/whatever, including that other partner
could dramatically change it's solution properties and be an easy fix for
your handling/formulation issues.
Good luck!
On Fri, Jul 14, 2017 at 6:33 AM,
Hi Chris,
In your Ni-NTA buffer, the total [Na+] could be greater than 530mM after
titration. Likely, your protein likes higher concentration salt for surface
charge stabilization. Adding Glycerol may further modify the charge
distribution and make the protein happy in the solution. For further
HEPES pH 7.5, 10% glycerol)
works. It should work well with crystallization.
Cheers,
Chun
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Chris Fage
Sent: Friday, July 14, 2017 3:33 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Protein rapidly precipitates when off ice
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Protein rapidly precipitates when off ice
Dear All,
Thank you for the many suggestions. After sending my first message to the BB, I
tried exchanging the sample into buffer containing 5 mM EDTA and also into
buffer at pH 9.0 (using BICINE). Neither
Dear All,
Thank you for the many suggestions. After sending my first message to the
BB, I tried exchanging the sample into buffer containing 5 mM EDTA and also
into buffer at pH 9.0 (using BICINE). Neither of these appear to have
helped the instability--precipitation still occurred within ~1 min
Dear all,
I will give my advice once again and hopefully you will discuss on it the
next 2 days :P
Chris might be worth trying a 2ndary or 3ary structure prediction tool to
ensure that you do not have f.i. flexible ends.
Disordered regions may lead to this kind of issues, as well.
Personally I
I'd try varying the pH independent of the theoretical pI, sometimes the real pI
is very different.
(I've worked on a protein with theoretical pI 9.2, real pI determined by
iso-electric focussing 7.8).
I'd also try limited proteolysis on the milky sample and see if you can
solubilise it while a
Hi,
I was in Sung-Hou Kim's group when this work below was performed and
published and I also tried it out on many occasions. Elegant piece of
work and certainly worth trying.
Aside from the suggestions of trying different pH and related optimum
solubility screening and if higher salt and
Hi Chris,
What is the theoretical pI of your protein? If it is around pH 7.5, you might
try gel filtering your protein into a different buffer/pH combination. Try
changing by at least 1 pH unit in either direction.
If the pI isn't a problem, then you might try try solubility screening as
