Dear Sankaranarayanan Srinivasan
Try replacing both glycerol and ethylene glycol by propylene glycol in part
or completely.
Enrico.
On Wed, 02 Jan 2013 18:59:21 +0100, Sankaranarayanan Srinivasan
<texs...@gmail.com> wrote:
Dear all,
A very happy new year to all.
I would appreciate some expert advice on optimizing a crystallization
condition in which the initial hits were obtained with ethylene glycol as
the main precipitant. Here is the summary of things tried.
We have a protein, size (31Kda) and the starting protein buffer is 0.1M
Tris pH7.5, 0.1M NaCl, 10% glycerol.
The initial crystal hit was obtained from the emerald cryo kit condition
that has 0.1M imidazole pH 8.0 , 50% (v/v) ethylene glycol. The crystals
were tiny (10-20um). A crystallization matrix to obtain better crystals
by
varying the imidazole pH and ethylene glycol concentrations was tried
from
which the best condition obtained was 0.1M imidazole pH 6.5 , 30% (v/v)
ethylene glycol. The crystals were slightly bigger 50um.
On trying the additive screen, bigger crystals (200um) were obtained, but
putting them under the x-ray beam with direct freezing did not yield any
diffraction spots.
Trying other cryo-conditions like glycerol and 50-50 paratone/oil mixture
also yielded similar results.
Low resolution spots near the beam stop were also not seen. Similarly
spots
indicative of salt was also not seen. It just had hazy ice rings kind of
stuff. (The beam was definitely on the crystal)
To check if what we have was salt, a control condition with no protein
was
tried. Also the crystals were run on a gel after thorough washing. Both
these tests, show that they are definitely protein crystals and not salt.
Seeding also did not yield any improved crystals.
I was suggested using di-ethylene glycol, propane diol as alternatives.
I would greatly appreciate if you can give your opinion on using other
di-alcohols as precipitants or other ways to improve these crystals.
I tried searching the PDB to see if someone had actually used ethylene
glycol as a precipitant, most of them were used as a cryo condition than
actually as a precipitant.
Thank you very much in advance.
Regards
Shankar Srinivasan
--
Enrico A. Stura D.Phil. (Oxon) , Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449 Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
