A less viscous oil used in small molecule low-temp work is PerFluoroPolyEther. Its general use is as an ultra-high vacuum pump oil, so it has an extremely low volatility. Additionally, PFPE has a very low fracture temperature and is non-reactive. I have used it in both small molecule and macromolecular projects.
Although sold by several companies, I buy it from Sigma-Aldrich. FOMBLIN R YVAC 14/6 Cat. # 317934-100g If you use this, note that it is slightly hygroscopic. Thus, I pull several 1ml aliquots from the bottle at a time and then reseal the bottle. And, as with many oils, be cautious using with membrane bound proteins...if you are unlucky, you may dissolve your crystal. Kris _____ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of David Briggs Sent: Monday, March 05, 2007 9:47 AM To: [email protected] Subject: [ccp4bb] How to get rid of Membrane formed on hanging droplets? Hi, What you describe is "skin" - rumoured to be denatured protein. http://xray.bmc.uu.se/~terese/crystallization/pics/SKIN2.JPE <http://xray.bmc.uu.se/%7Eterese/crystallization/pics/SKIN2.JPE> And it's a pain, especially when your crystals stick to it. Best suggestions are: 1) Try screening additives or detergents that might stabilise it 2) Try changing the crystallisation setup to microbatch under oil - I have never had skin that way. If you can't get rid of the skin, you're going to have to excise your crystals out very carefully. This has worked for me in the past: 1) Put a big blob (~10-20ul) of 50:50 Paratone oil : Mineral Oil on top of the drop. This does two things - prevents the drop drying out as your wrestle away the skin and can be your cryoprotectant as well. 2) Use a fine accupuncture needle or hampton probe to cut the patch of skin away with your crystal attached. 3) manoeuvre the crystal+skin away from the drop (but still in the oil) and then very very very carefully start trying to pull the skin away with the probe or a loop (or even both - one in each hand) 4) The Oil will give you plenty of time to play with your crystal to try and get the skin off. By the time you've done this you will also have got most excess mother liquor away so you can go right ahead and freeze your crystal. This requires patience, but the more skin you get away the better the freeze and the less you will harm your diffraction. It goes without saying that if you can get a big xtal NOT attached to the skin, then life is much easier. Good Luck! Dave On 05/03/07, Wang, Yeming (NIH/NIEHS) [F] < [EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]> > wrote: Dear everyone, I am working on crystallization of a protein/RNA complex recently. The crystals were initially grown from BICINE( 9.0) 0.1M, 1,4-Dioxane 2%(v/v) , PEG 20,000 10%(w/v), at 10mg/ml. I noticed that there was a membrane formed on the surface of the hanging droplets. This membrane seems very sticky. Consequently, almost all of the crystals stick to this membrane and can't be seperated for data collection. Sitting drops were also tried but crystals stick to the bottom of the sitting well. Different buffer(Tris, CHES), different PEG(PEG 8000, PEG3350, from 10%-1%) and different protein concentration (10-3mg/ml) were also tried, but the sticky membrane was still there. Does anyone have some experience solving this problem? Any suggestions would be highly appreciated! Yeming --------------------- Yeming Wang, Ph.D. Laboratory of Structural Biology: Macromolecular Structure Group National Institute of Environmental Health Sciences National Institute of Health Mailing Address: Street Address: NIEHS, MD F3-05 NIEHS, Building 101, Room F363 P.O. BOX 12233 111 T.W. Alexander Drive RTP, NC 27709 RTP, NC 27709 Tel (o): 919-316-4634 E-mail: [EMAIL PROTECTED] -- --------------------------------------- David Briggs, PhD. Father & Crystallographer www.dbriggs.talktalk.net <http://www.dbriggs.talktalk.net> iChat AIM ID: DBassophile --------------------------------------- Anyone who is capable of getting themselves made President should on no account be allowed to do the job. - Douglas Adams
