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[ccp4bb] How to get rid of Membrane formed on hanging droplets? |
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CCP4bb navigationCCP4bb <-- 2007 <-- March 2007 <-- 05 March 2007Subject: How to get rid of Membrane formed on hanging droplets? From: David Briggs bassophile {- at -} GMAIL {- dot -} COM Date: 2007-03-05 What you describe is "skin" - rumoured to be denatured protein. http://xray.bmc.uu.se/~terese/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] > > 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: wangy2@niehs.nih.gov > -- --------------------------------------- David Briggs, PhD. Father & Crystallographer 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 CCP4bb navigationCCP4bb <-- 2007 <-- March 2007 <-- 05 March 2007 |
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