Re: [ccp4bb] Problems in crystallization
I've seen this effect too. In the case I saw the drops were growing (and spreading) due to a reverse diffusion gradient. Optimised conditions used "salting in" method i.e. crystallising as the [pptnt] decreases during equilibration due to dilution as water is Tx from well to drop. Rare but not unknown. David Hargreaves Associate Principal Scientist _ AstraZeneca DECS, CP&SS Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF Tel +44 (0)01625 518521 Fax +44 (0) 1625 232693 David.Hargreaves @astrazeneca.com Please consider the environment before printing this e-mail -- AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking Compliance with our Code of Conduct and Policies. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Eva Bligt-Lindén Sent: 07 November 2012 12:07 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Problems in crystallization Dear ccp4 users, I have a problem in the crystallization of my target protein. Whenever I set up a vapour diffusion experiment, either hanging or sitting drops, the drops spread out. The surface tension is completely lost in 80-90% of the droplets. Have any one experienced something similar? What could be the reason for this strange behaviour? I have tried three different commercial screens with 96 condition each and there is no difference between the screens. There is no difference between manual or robotic setups either. The protein buffer is 40 mM Tris, 2 mM MgCl2 buffer, pH 7.4. The buffer controls are all ok. Kind regards, Eva Eva Bligt-Lindén (M.Sc.) PhD student Structural Bioinformatics Laboratory Department of Biosciences, Åbo Akademi University BioCity, Tykistökatu 6A FI-20520 Turku Finland
Re: [ccp4bb] Problems in crystallization
On Wed, 2012-11-07 at 16:04 +0200, Eva Bligt-Lindén wrote: > To my knowledge the protein is not a > surface-tension-reducing-protein and there is no detergent in the > sample. However your observations indicate that your sample has reduced surface tension. When you say that "to your knowledge it is not", do you mean that you have some other data that contradicts such conclusion? -- "I'd jump in myself, if I weren't so good at whistling." Julian, King of Lemurs
Re: [ccp4bb] Problems in crystallization
Eva Yes batch under oil will solve the problem. We regularly used to crystallize conditions with high levels of MPD under oil without difficulty. I agree that the protein might be reducing the surface tension, but I've never heard of such a strong effect. Maybe the protein is unstable and "wants" to denature onto the surface of the plate. Is it possible that you have any low-molecular-weight organics in your protein sample? You could try the "UV-compatible" versions of crystallization plates because they're more hydrophobic than the regular PS ones. The polypropylene ones are even more hydrophobic, similar to siliconized glass. Salt precipitants will tend to reduce drop spreading, PEG to increase it. It's partly a matter of surface tensions, partly of the affinity of the ingredients for the plastic/glass. The air-liquid interface energy competes with the liquid-solid interface energy. Patrick On 7 November 2012 13:41, anna anna wrote: > Why don't you try batch under oil? > > 2012/11/7 Eva Bligt-Lindén > >> Dear ccp4 users, >> >> I have a problem in the crystallization of my target protein. Whenever I >> set up a vapour diffusion experiment, either hanging or sitting drops, the >> drops spread out. The surface tension is completely lost in 80-90% of the >> droplets. Have any one experienced something similar? What could be the >> reason for this strange behaviour? I have tried three different commercial >> screens with 96 condition each and there is no difference between the >> screens. There is no difference between manual or robotic setups either. >> The protein buffer is 40 mM Tris, 2 mM MgCl2 buffer, pH 7.4. The buffer >> controls are all ok. >> >> Kind regards, >> Eva >> >> __**__ >> >> Eva Bligt-Lindén (M.Sc.) >> PhD student >> Structural Bioinformatics Laboratory >> >> Department of Biosciences, >> Åbo Akademi University >> BioCity, Tykistökatu 6A >> FI-20520 Turku >> Finland >> > > -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Problems in crystallization
Dear Eva, are you using siliconized cover slips or normal ones ? Protein drops on not siliconized glass tend to spread especially if they contain alcohol or detergent. But this shouldn't be a problem with sitting drops usually. Good luck ! Ulrike
Re: [ccp4bb] Problems in crystallization
Those are the EasyXtal 15 well tools with the EasyXtal DG-Crystal Supports. Look at this link to find them: http://www.qiagen.com/products/protein/crystallization/default.aspx I agree that they keep the drops from spreading out, but I have experienced trouble harvesting smaller crystals from these xtal tools. Good luck! Bryan From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Rob Gillespie Sent: Wednesday, November 07, 2012 9:19 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Problems in crystallization Qiagen used to sell hanging-drop trays with screw-cap tops, and the tops had six rings on them (3 large, 3 small). The rings had a lip on them that kept the drop from spreading beyond the edge. I found these quite useful when setting up drops that had detergent in them. I unfortunately don't know what they are called, as I have several at my bench but not the packaging they arrived in. On Wed, Nov 7, 2012 at 9:04 AM, Eva Bligt-Lindén wrote: Thank you for your replies. To my knowledge the protein is not a surface-tension-reducing-protein and there is no detergent in the sample. I have tried different plates and still the same result. I will try a different crystallization technique such as batch under oil or counter-diffusion if I do not solve this problem. But now I am curious to know what is causing the disturbed surface tension and if someone else has experienced and dealt with something similar. Kind regards, Eva Quoting anna anna : Why don't you try batch under oil? 2012/11/7 Eva Bligt-Lindén Dear ccp4 users, I have a problem in the crystallization of my target protein. Whenever I set up a vapour diffusion experiment, either hanging or sitting drops, the drops spread out. The surface tension is completely lost in 80-90% of the droplets. Have any one experienced something similar? What could be the reason for this strange behaviour? I have tried three different commercial screens with 96 condition each and there is no difference between the screens. There is no difference between manual or robotic setups either. The protein buffer is 40 mM Tris, 2 mM MgCl2 buffer, pH 7.4. The buffer controls are all ok. Kind regards, Eva __**__ Eva Bligt-Lindén (M.Sc.) PhD student Structural Bioinformatics Laboratory Department of Biosciences, Åbo Akademi University BioCity, Tykistökatu 6A FI-20520 Turku Finland Eva Bligt-Lindén (M.Sc.) PhD student Structural Bioinformatics Laboratory Department of Biosciences, Åbo Akademi University BioCity, Tykistökatu 6A FI-20520 Turku Finland -- Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] Problems in crystallization
Qiagen used to sell hanging-drop trays with screw-cap tops, and the tops had six rings on them (3 large, 3 small). The rings had a lip on them that kept the drop from spreading beyond the edge. I found these quite useful when setting up drops that had detergent in them. I unfortunately don't know what they are called, as I have several at my bench but not the packaging they arrived in. On Wed, Nov 7, 2012 at 9:04 AM, Eva Bligt-Lindén wrote: > Thank you for your replies. To my knowledge the protein is not a > surface-tension-reducing-**protein and there is no detergent in the > sample. I have tried different plates and still the same result. I will try > a different crystallization technique such as batch under oil or > counter-diffusion if I do not solve this problem. But now I am curious to > know what is causing the disturbed surface tension and if someone else has > experienced and dealt with something similar. > > Kind regards, > Eva > > > Quoting anna anna : > > Why don't you try batch under oil? >> >> 2012/11/7 Eva Bligt-Lindén >> >> Dear ccp4 users, >>> >>> I have a problem in the crystallization of my target protein. Whenever I >>> set up a vapour diffusion experiment, either hanging or sitting drops, >>> the >>> drops spread out. The surface tension is completely lost in 80-90% of the >>> droplets. Have any one experienced something similar? What could be the >>> reason for this strange behaviour? I have tried three different >>> commercial >>> screens with 96 condition each and there is no difference between the >>> screens. There is no difference between manual or robotic setups either. >>> The protein buffer is 40 mM Tris, 2 mM MgCl2 buffer, pH 7.4. The buffer >>> controls are all ok. >>> >>> Kind regards, >>> Eva >>> >>> ____ >>> >>> >>> Eva Bligt-Lindén (M.Sc.) >>> PhD student >>> Structural Bioinformatics Laboratory >>> >>> Department of Biosciences, >>> Åbo Akademi University >>> BioCity, Tykistökatu 6A >>> FI-20520 Turku >>> Finland >>> >>> >> > > > __**__ > > Eva Bligt-Lindén (M.Sc.) > PhD student > Structural Bioinformatics Laboratory > > Department of Biosciences, > Åbo Akademi University > BioCity, Tykistökatu 6A > FI-20520 Turku > Finland >
Re: [ccp4bb] Problems in crystallization
Thank you for your replies. To my knowledge the protein is not a surface-tension-reducing-protein and there is no detergent in the sample. I have tried different plates and still the same result. I will try a different crystallization technique such as batch under oil or counter-diffusion if I do not solve this problem. But now I am curious to know what is causing the disturbed surface tension and if someone else has experienced and dealt with something similar. Kind regards, Eva Quoting anna anna : Why don't you try batch under oil? 2012/11/7 Eva Bligt-Lindén Dear ccp4 users, I have a problem in the crystallization of my target protein. Whenever I set up a vapour diffusion experiment, either hanging or sitting drops, the drops spread out. The surface tension is completely lost in 80-90% of the droplets. Have any one experienced something similar? What could be the reason for this strange behaviour? I have tried three different commercial screens with 96 condition each and there is no difference between the screens. There is no difference between manual or robotic setups either. The protein buffer is 40 mM Tris, 2 mM MgCl2 buffer, pH 7.4. The buffer controls are all ok. Kind regards, Eva __**__ Eva Bligt-Lindén (M.Sc.) PhD student Structural Bioinformatics Laboratory Department of Biosciences, Åbo Akademi University BioCity, Tykistökatu 6A FI-20520 Turku Finland Eva Bligt-Lindén (M.Sc.) PhD student Structural Bioinformatics Laboratory Department of Biosciences, Åbo Akademi University BioCity, Tykistökatu 6A FI-20520 Turku Finland
Re: [ccp4bb] Problems in crystallization
Why don't you try batch under oil? 2012/11/7 Eva Bligt-Lindén > Dear ccp4 users, > > I have a problem in the crystallization of my target protein. Whenever I > set up a vapour diffusion experiment, either hanging or sitting drops, the > drops spread out. The surface tension is completely lost in 80-90% of the > droplets. Have any one experienced something similar? What could be the > reason for this strange behaviour? I have tried three different commercial > screens with 96 condition each and there is no difference between the > screens. There is no difference between manual or robotic setups either. > The protein buffer is 40 mM Tris, 2 mM MgCl2 buffer, pH 7.4. The buffer > controls are all ok. > > Kind regards, > Eva > > __**__ > > Eva Bligt-Lindén (M.Sc.) > PhD student > Structural Bioinformatics Laboratory > > Department of Biosciences, > Åbo Akademi University > BioCity, Tykistökatu 6A > FI-20520 Turku > Finland >
Re: [ccp4bb] Problems in crystallization
Hi there, You could try to go for liquid-liquid interface crystallisation (in capillaries). The minimum volume would be no less than 1.5 microl protein + 1.5 microl precipitant, i.e. not the volumes used by nanodrop robots. Crystallisation under oil (or similar liquids) should also have the same effect (no spreading out). HTH, Fred. On 07/11/12 13:07, Eva Bligt-Lindén wrote: Dear ccp4 users, I have a problem in the crystallization of my target protein. Whenever I set up a vapour diffusion experiment, either hanging or sitting drops, the drops spread out. The surface tension is completely lost in 80-90% of the droplets. Have any one experienced something similar? What could be the reason for this strange behaviour? I have tried three different commercial screens with 96 condition each and there is no difference between the screens. There is no difference between manual or robotic setups either. The protein buffer is 40 mM Tris, 2 mM MgCl2 buffer, pH 7.4. The buffer controls are all ok. Kind regards, Eva Eva Bligt-Lindén (M.Sc.) PhD student Structural Bioinformatics Laboratory Department of Biosciences, Åbo Akademi University BioCity, Tykistökatu 6A FI-20520 Turku Finland -- Fred. Vellieux (B.Sc., Ph.D., hdr) IBS / ELMA 41 rue Jules Horowitz F-38027 Grenoble Cedex 01 Tel: +33 438789605 Fax: +33 438785494
Re: [ccp4bb] Problems in crystallization
is your protein a STRP (surface-tension-reducing protein)? Seriously, did you try different plates? Old-fashioned siliconised cover slips on Linbro plates? Does your sample contain detergent? Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 7 Nov 2012, at 13:07, Eva Bligt-Lindén wrote: > Dear ccp4 users, > > I have a problem in the crystallization of my target protein. Whenever I set > up a vapour diffusion experiment, either hanging or sitting drops, the drops > spread out. The surface tension is completely lost in 80-90% of the droplets. > Have any one experienced something similar? What could be the reason for this > strange behaviour? I have tried three different commercial screens with 96 > condition each and there is no difference between the screens. There is no > difference between manual or robotic setups either. The protein buffer is 40 > mM Tris, 2 mM MgCl2 buffer, pH 7.4. The buffer controls are all ok. > > Kind regards, > Eva > > > > Eva Bligt-Lindén (M.Sc.) > PhD student > Structural Bioinformatics Laboratory > > Department of Biosciences, > Åbo Akademi University > BioCity, Tykistökatu 6A > FI-20520 Turku > Finland
