How do you know your precipitate is AmSO4? At the low concentration youre using, my guess is that the precipitate is protein. It doesnt matter whether AmSo4 is a cryoprotectant or not (at 0.2 M, its not), you have more than enough MPD and PEG400 in your solution to do the job.
Have you looked at diffraction patterns yet? The best (and really only) way to see if the cryoprotection worked is to see whether there are ice-rings. Unless youre freezing in the wrong way, the only issue I can think of that would make your condition not suitable for direct freezing is if the volume of the drop has increased during equilibration, so that the MPD concentration is now much less than 30-40% (for example by having glycerol in the sample but not in the screen solution). Hope this helps, Bert Bert van den Berg University of Massachusetts Medical School Program in Molecular Medicine Biotech II, 373 Plantation Street, Suite 115 Worcester MA 01605 Phone: 508 856 1201 (office); 508 856 1211 (lab) e-mail: bert.vandenb...@umassmed.edu http://www.umassmed.edu/pmm/faculty/vandenberg.cfm -----Original Message----- From: HanJie_HCT Tai [mailto:chemtai2...@hotmail.com] Sent: Fri 4/3/2009 4:10 PM To: Van Den Berg, Bert; ccp4bb@jiscmail.ac.uk Subject: RE: [ccp4bb] How to loop up crystal from AmSO4. 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! <http://ie8.msn.com/microsoft/internet-explorer-8/en-us/ie8.aspx?ocid=B037MSN55C0701A>
