SnowDeer,
Additives are indeed a good idea, make sure you do it in an informed
manner, each additive is different and you will
get a benefit only if you know how to do it right. I do not recommend a
random approach.
For example, you mention glycerol. There is a lot to know on the subject
and probably more to discover.
Glycerol will help freezing/thawing cycles and improve protein stability
if you want to work at higher temperatures than
4°C. You should check increased stability effect out since it is easier to
work outside a cold room.
I assume you are trying to set up your drops at the same temperature at
which they will equilibrate. Which is a good thing to do.
Yet, plan your experiments carefully. Also look at previous messages on
ccp4bb on the subject of glycerol
in particular regarding propanediol. Annie Hassell among others has posted
some very good comments. This bullettin board
has been very keen on the subject in the past, so you can learn a lot from
the archives.
Glycerol is also great to reduce nucleation. If you decide to add glycerol
to the protein solution (for solubility, but in your case it might be for
stability
reasons), you also need to have a higher (double) glycerol concentration
in the reservoir else you will risk finding that your drops will get
biggger and
not smaller. This note of caution applies to vapour diffusion set ups as
equilibration can be tricky in such context:
Vera,L., Czarny, B., Georgiadis, D., Dive, V., Stura, E.A. (2011)
Practical Use of Glycerol in Protein Crystallization. Cryst. Growth & Des.
11 :2755–2762.
http://pubs.acs.org/doi/abs/10.1021/cg101364m
Good luck, but most important work with precision. To go from small to big
crystals you need to be very
precise on how you set up your experiments and understand the rate at
which you equilibrate your drops.
Enrico.
On Thu, 08 Sep 2011 09:52:40 +0200, SnowDeer <[email protected]> wrote:
To Boaz: I seperated the large crystals and checked its diffraction
already.
While the diffraction is quite poor, only several dots could be seen. T_T
I washed the seeds twice with my buffer and seed the drop immediately
after
setting it. Thanks for your advices and I will try the additives.
To Charles, Enrico, Bernie & Tiantian: Thanks for your kindly advices, I
set
different conditions for the buffers with glycerol and different
protein/reservoir volume ratios already following your instructions. :)
To David: Hmm...my crystals are smaller than the smallest loop T_T. It's
quite hard for me to pick them up (due to my clumsy fingers...lol).
Thanks
for your advices and the review.
I have another question: I usually stored my protein samples aliquots at
-80
degree and thaw the small aliquots when I need to use. While my senior
said
it will harm the protein so she suggested to keep them at 4 degree. So
it is
possible that I got the small crystals coz the freezing and thawing alter
the proteins?
Thanks very much.
SnowDeer
--
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: [email protected] Fax: 33 (0)1 69 08 90 71
