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
<[log in to unmask]> 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: [log in to unmask] Fax: 33 (0)1 69 08 90 71
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