We rarely use glycerol anymore, because it seems to fail so often for
many of our current proteins. Try glucose, 25-30%. This is most
conveniently done by weighing 125-150 mg of glucose in a microcentrifuge
tube, then addding well solution to the 0.5 mL mark and mixing until
completely dissolved. Then you can try dunking crystals in the cryo
solution, or, you can try the "no-fail" method (which does fail on
occasion) of cryoprotecting directly in the crystallization drop by slow
addition of the cryoprotectant. See
http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Mounting+Protein+Crystals.
We have often found the slow addition of a glucose cryoprotectant works
for fragile, high solvent content crystals that are prone to cracking
under osmotic stress.
Other alternatives include high concentrations of sodium malonate
(1-2M), or high concentrations of sodium formate (I think around 4 M?).
These could also be introduced gradually if required.
Good luck!
_______________________________________
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu
On 5/4/2015 2:43 PM, Faisal Tarique wrote:
Hello everyone
Can anybody suggest me a cryo condition for a crystal obtained in
MIDAS screen of Molecular Dimension:
G1> 0.1MTris8.0G10.1Mpotassium chloride25% v/vSOKALAN®CP7 0.1MHEPES7.0
G2>0.3Mammonium formate20% v/vSOKALAN® CP 5 0.1MHEPES7.0
Crystals are in beautiful cuboid shaped but all sorts of PEG
combinations and Glycerol formulation failed to prevent it from
cracking and dissolving.
Has anybody faced a similar situation as mentioned above and what
precaution was taken to prevent it from cracking or dissolving.
Your suggestions will be of immense help
Thanks in advance
