Mahesh,
Try 25-30% glucose. You can gradually add well solution + 37.5-40%
glucose directly to the drop if your crystals are sensitive to changes
in osmotic pressure. If your drop is evaporating too quickly, try
working in the cold room or under oil to slow down evaporation. You can
find our standard cryoprotection protocol on our wiki here
<http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Mounting+Protein+Crystals>
under "No-fail" cryoprotection.
Cheers,
_______________________________________
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 1/22/2014 12:28 PM, Mahesh Lingaraju wrote:
Hello folks,
I have crystals for a protein which form in 0.1 M MES pH 6-6.75, 10%
dioxane and 1.6-2 M Ammonium sulphate. Based on little experience I
have finding the right cryoprotection for salt based conditions; I
have tried glycerol (15-25%), Ethylene glycol (20-25%), DMSO (15-20%)
and increasing ammonium sulphate concentration in presence of 5-10%
glycerol. The crystals disintegrate in any kind of PEG based cryo. I
made all these solutions in the mother liquor and tested if they
freeze clearly before using them. However when I loop the crystals and
try to soak them in these cryo- mother liquor, a lot of salt crystals
suddenly form around the protein crystal and I see diffraction only
from these salt crystals. The best I have been able to get so far is ~
9Å diffraction (Home-source) with DMSO as the cryoprotectant.
I have also tried using 1 M sodium malonate as the cryoprotectant but
my crystals are not too stable in this mother liquor probably because
I had to lower the ammonium sulphate by ~ 7-10% to make the drop not
form salt crystals instantly when exposed to air.
Other than trying to make the crystals more cryo-ready by finding
other hits or may be growing the same crystals with some
cryoprotectant, I was wondering if any of you have ideas based on your
experience or suggestions in case I am doing everything wrong in the
first place.
Thanks for all the help,
Mahesh
