What about nature's favourite cryoprotectant, trehalose? ________________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Roger Rowlett <rrowl...@colgate.edu> Sent: Tuesday, 5 May 2015 7:22 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] cryo condition
We rarely use glycerol anymore, because it seems to fail so often for many of our current proteins. Try glucose, 25-30%. This is most conveniently done by weighing 125-150 mg of glucose in a microcentrifuge tube, then addding well solution to the 0.5 mL mark and mixing until completely dissolved. Then you can try dunking crystals in the cryo solution, or, you can try the "no-fail" method (which does fail on occasion) of cryoprotecting directly in the crystallization drop by slow addition of the cryoprotectant. See http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Mounting+Protein+Crystals. We have often found the slow addition of a glucose cryoprotectant works for fragile, high solvent content crystals that are prone to cracking under osmotic stress. Other alternatives include high concentrations of sodium malonate (1-2M), or high concentrations of sodium formate (I think around 4 M?). These could also be introduced gradually if required. Good luck! _______________________________________ 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 5/4/2015 2:43 PM, Faisal Tarique wrote: > Hello everyone > > Can anybody suggest me a cryo condition for a crystal obtained in > MIDAS screen of Molecular Dimension: > > G1> 0.1MTris8.0G10.1Mpotassium chloride25% v/vSOKALAN®CP7 0.1MHEPES7.0 > > G2>0.3Mammonium formate20% v/vSOKALAN® CP 5 0.1MHEPES7.0 > > Crystals are in beautiful cuboid shaped but all sorts of PEG > combinations and Glycerol formulation failed to prevent it from > cracking and dissolving. > > Has anybody faced a similar situation as mentioned above and what > precaution was taken to prevent it from cracking or dissolving. > > Your suggestions will be of immense help > > Thanks in advance
