Re: [ccp4bb] How to loop up crystal from AmSO4.

[Roger Rowlett]

I would think 30% MPD or more should normally be enough to cryoprotect. If that is not the case, then you can always supplement with additional MPD, glycerol, or glucose. If I understand correctly, your crystals are forming in the presence of some additional protein precipitate? If so, you might try transferring crystals and precipitate to fresh mother liquor and gently teasing away the precipitate. I gather that your crystals do not grow at a lower precipitant concentration, where precipitate would be less or non-existent? It often takes longer to get crystals this way, but they are also often larger and free of precipitate. We had two proteins that took 3 weeks and 3 months to crystallize, respectively, in large, clean form. (You can see the 3-month crystals on the Hampton Research web site as the image next to "Cryocrystallography" on the products page. Cheers, -- Roger S. Rowlett Professor Colgate University Presidential Scholar 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...@mail.colgate.edu HanJie_HCT Tai wrote: In the drop, the amSO4 becomes/casuses precipitate (I guess). The crystals were grown from the precipitate within a few days. When I looped up the crystal the precipitate was also looped up.   And I don't know whether AmSO4 is a cryoprotectant or not. When I freeze the crystal in LN, the crystal is not in a glassy drop under the microscope. Rgds, (HanJie)   Date: Fri, 3 Apr 2009 13:45:40 -0400 From: lambertus.vandenb...@umassmed.edu Subject: Re: [ccp4bb] How to loop up crystal from AmSO4. To: CCP4BB@JISCMAIL.AC.UK I also don't understand what you mean by "The crystal always sticks with AmSO4" and "the crystal was still surrounded by AmSO4".... Is there solid AmSO4 in the drop? Judging from the concentration you state, this can't be the case. Just loop up the crystal and freeze I would say, the MPD + PEG400 should be plenty high enough for good cryo. Bert van den Berg University of Massachusetts Medical School -----Original Message----- From: CCP4 bulletin board on behalf of James Holton Sent: Fri 4/3/2009 1:36 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] How to loop up crystal from AmSO4. If you want to get rid of mother liquid around crystal, mount it in oil. Howerver, what is wrong with NH4SO4?  If your crystal can tolerate near-saturated NH4SO4, it is a cryoprotectant all by itself. -James Holton MAD Scientist HanJie_HCT Tai wrote: > Hi, >  > I have a protein-DNA complex grown in 30 -40% MPD/0.2M Ammonium > sulfate/5% PEG 400/ 0.1M Tris (8.5). >  > I believe that 30%MPD above does not require any cryoprotectant but i > hv a problem to loop a crystal up. The crystal always sticks with AmSO4. >  > I tried to add 2ul up to 6ul reservoir solution into the protein drop > (1+1 ul). Although AmSO4 was diluted, the crystal was still surrounded > by AmSO4. >  > The loop size is 0.1 -0.2mm, the crystal is about 0.1 mm. >  > I tried to grow crystal without AMSO4 or 0.1M AmSO4, but no crystals > was shown up. >  > Any suggestion to loop up the crystal without any AMSO4 attached? > > Regards, > HCT (HanJie) > > > > ------------------------------------------------------------------------ > Rediscover Hotmail®: Get quick friend updates right in your inbox. > Check it out. > <http://windowslive.com/RediscoverHotmail?ocid=TXT_TAGLM_WL_HM_Rediscover_Updates1_042009> Quick access to your favorite MSN content and Windows Live with Internet Explorer 8. Download FREE now!