Several have mentioned harvesting in the cold room to reduce evaporation.  I used to do this also as a postdoc, but I worried whether I risked nitrogen gas poisoning from liquid N2 boil-off, since the cold room did not seem very well-ventilated.  I’ve also hesitated to recommend it to trainees in my current lab for the same reason.  Does anyone have solid information on this?  I would like to be convinced that such fears are unfounded …

 

Evette S. Radisky, Ph.D.
Assistant Professor
Mayo Clinic Cancer Center
Griffin Cancer Research Building, Rm 310
4500 San Pablo Road
Jacksonville, FL 32224
(904) 953-6372

From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Roger Rowlett
Sent: Thursday, July 12, 2012 2:11 PM
To: [log in to unmask]
Subject: Re: [ccp4bb] cryo for high salt crystal

 

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: [log in to unmask]

 

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

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From: [log in to unmask]
To: [log in to unmask]; [log in to unmask]
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

 

 

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From: CCP4 bulletin board [[log in to unmask]] on behalf of m zhang [[log in to unmask]]
Sent: Tuesday, July 10, 2012 11:28 AM
To: [log in to unmask]
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