We frequently crystallize one of our proteins and variants of it in
1.6-1.8 M ammonium sulfate solutions. Cryoprotection with 25-30%
glycerol or 25-30% glucose does not cause precipitation of salts. Both
KCl (4.6 M) and ammonium sulfate (5.6 M) have enormous solubilities in
water, so I would not expect cryoprotectant concentrations of glycerol
or glucose to cause precipitation (We can save cryoprotectant solutions
of at least 2 M ammonium sulfate indefinitely). How are you introducing
cryprotectant? We use one of two methods:
1. Fish the crystal out of the mother liquor and place into artificial
mother liquor with the same composition as the well solution +
cryoprotectant. For glycerol or other liquids, you have to make this
from scratch. For glucose, we just weigh out 300 mg of glucose in a
microcentrifuge tube and make to the 1.0 mL mark with well solution.
(Mix well of course before use. Gentle heating in a block or
sonication will help dissolve the glucose.
2. Add 4 volumes of artificial mother liquor + 37.5% cryoprotectant to
the drop the crystals are in. You can do this all at once, or in
stages, keeping the drop hydrated by placing the hanging drop back
in the well between additions.
If your drops are drying out during crystal harvesting (very possible in
dry conditions), you might try harvesting in the cold room, where
evaporation is slower. We often have problems with crystal cracking and
drop-drying in the winter months when the humidity is very low indoors.
The cold room is usually humid enough and cold enough to slow
evaporation to allow crystal harvesting. (I hate working in the meat
locker, though.)
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 7/12/2012 12:55 PM, m zhang wrote:
Hi Jim,
25% is w/v. Thanks for the information. Will check the webinar.
Thanks,
Min
------------------------------------------------------------------------
From: jim.pflugr...@rigaku.com
To: mzhang...@hotmail.com; CCP4BB@JISCMAIL.AC.UK
Subject: RE: [ccp4bb] cryo for high salt crystal
Date: Tue, 10 Jul 2012 17:39:56 +0000
Sucrose, sorbitol, Splenda, trehalose, etc, but instead of 25% (is
that w/v or v/w?), try using 100% saturated in reservoir, 75%
saturated in reservoir, or 50% saturated in reservoir. You will have
to TEST these. See also this webinar on cryocrystallography which
shows how to make these solutions: http://www.rigaku.com/node/1388
You could also try high salt solutions with similar technique.
Good luck!
Jim
------------------------------------------------------------------------
*From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of m
zhang [mzhang...@hotmail.com]
*Sent:* Tuesday, July 10, 2012 11:28 AM
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* [ccp4bb] cryo for high salt crystal
regaentDear All,
I am sure this question was discussed before. But I am wondering if
anyone got the same experience as I do.
I got a crystal out of condition with 1M KCl, 1.4M Ammonium sulfate at
pH7. I tried to use glycerol, ethylene glycol, 25% sucrose, paraton-N
oil, or ammonium sulfate itself: The problem is that all the cryo plus
original reagents in the reservoir precipitate the salts out. And more
serious problem is because of high salt in the condition, while I am
trying to loop the crystal, both the drop and cryoprotectant drop form
salt crystals (not sure it is KCl or ammonia sulfate) significantly
and very quickly, that cause my crystal dissolved. My crystal doesn't
seem to survive paraton-N oil. Does anyone here have similiar case?
any suggestion will be appreciated.
Thanks,
Min
