I would definitely try gelfiltration (how do you get rid of the cleaved tag
anyway, sample buffer exchange?) but especially ion exchange. A homogeneous
sample on SDS-PAGE and/or gelfiltration is often not (at all) homogeneous in
ion exchange. Beyond that i would make some point mutations on surface residues
and focus on those (ie try the surface entropy method).
Good luck, Bert
________________________________________
From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of eswar reddy
[eswar.uo...@gmail.com]
Sent: Friday, August 26, 2011 6:56 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Crystals with Organic solvents
Dear All
I was working on a Human protein and expression and
solubility is good in E.coli and purification is One step (His-Tag), and i
need to cleave the Histag before screens, if not the protein will precipitated
and Aggregated, but after trying for 1.2 years i have crystals and they are
with Organic solvents, (10 conditions), these crystals are inter grown like
broccoli shaped and i tried seeding, but it is not successful, and even i
tried with additive screen but the result is the same .... is there is any
idea to increase the size and shape of my protein crystals.
Any suggestions will be helpful for me
Thanks in Advance
