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You probably already know this, but nitrogen is not at all poisonous--about 78% of the air is nitrogen. I guess you were probably worried about asphyxiation....? JPK On Fri, Jul 13, 2012 at 4:19 PM, Radisky, Evette S., Ph.D. < [log in to unmask]> wrote: > 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**** > ------------------------------ > > 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**** > > ** ** > > ** ** > ------------------------------ > > *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**** > > ** ** > -- ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: [log in to unmask] *******************************************