Re: [ccp4bb] Crystallization of low solubility proteins from glycerol-containing solutions
Dear Roger, We have had success using the non-detergent sulphobetaines (NDSBs) to improve solubility of protein samples. For a couple of projects they have proved crucial for concentrating the protein to a reasonable level for crystallization (e.g. PMID: 18765907). Be careful since there is a big variation in price dependent on which particular one you use. Use them at ~200-300 mM and you may even be able to get rid of the glycerol. You may have to incubate the protein with the NDSBs overnight before concentration to get the full effect. Good luck Tom Walter Original message Date: Wed, 25 Aug 2010 10:19:13 -0400 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Roger Rowlett rrowl...@colgate.edu) Subject: [ccp4bb] Crystallization of low solubility proteins from glycerol-containing solutions To: CCP4BB@JISCMAIL.AC.UK Does anyone have practical experience crystallizing low solubility proteins from solutions containing significant (10-20%) glycerol? We can get small crystals by mixing 4:1 ratios of protein to well solution, but the drops do not concentrate back to the well solution volume as anticipated, even if the well solution is brought to 10-20% glycerol as well to balance osmolarity. Concentration of the protein further to reduce the protein:well solution ratio may not be practical (it crashes out) even in 20% glycerol. Unfortunately, glycerol seems to be required to maintain protein solubility, so that may not be practical to remove either. One thought is to add additional osmolyte to the well solution to draw down the drop volume once small crystals form, a kind of a macro-seeding approach, but I am not aware of a systematic way of doing this. Anyway, I am almost certain I am trying to re-invent the wheel,as someone has probably done something similar. Any suggestions would be appreciated. Cheers, -- -- Roger S. Rowlett 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
Re: [ccp4bb] problem of crystallization
A few ideas: 1) go even higher with protein concentration. Some ultra-soluble proteins may need 100 mg/mL. You may have to set up drops at higher ratios of protein:precipitant to achieve this. e.g. 3:1 or more. Use a test such as the Hampton PCT to tell you when you are in the right concentration range. 2) reductive methylation of lysine residues as Stephen suggested. This often reduces the solubility of proteins and will change the surface properties. 3) get rid of any glycosylation as the surface sugars increase solubility (and are flexible anyway and so bad for crystallization) 4) check the few drops which have precipitate and look for common factor e.g. 2M Ammonium Sulphate. You could then add a small amount of this ingredient (e.g. 100mM Ammonium Sulphate) to your protein and then re-screen. 5) Wait a bit longer. One week is still early days. Good luck Tom ** Tom Walter B.Sc. M.Res. ** ** Oxford Protein Production FacilityTel: +44 (0)1865 287747 ** ** Wellcome Trust Centre for Human Genetics Fax: +44 (0)1865 287547 ** ** Roosevelt Drive [EMAIL PROTECTED] ** ** Headington, Oxford OX3 7BNhttp://www.oppf.ox.ac.uk ** Original message Date: Tue, 13 May 2008 11:16:41 -0500 From: Jennifer Han-Chun Tsai [EMAIL PROTECTED] Subject: [ccp4bb] problem of crystallization To: CCP4BB@JISCMAIL.AC.UK Hi, This topic is not related to CCP4. I am having problem of crystallizing one protein. It's a pretty small protein with size around 15kDa. I have stock concentration around 100mg/mL. Crystallization plates I set up are with concentration of 10mg/mL, 30mg/mL and 50mg/mL. All the plates had been set up at least one week. Only around 5 wells per plate or less formed precipitation. The rest of wells are pretty clear still. Is there any suggestion for reducing protein solubility or increasing the chance of getting crystals? Thanks for your time, Jennifer
Re: [ccp4bb] Fwd: [ccp4bb] crystallisation robot
We use Cartesian Honeybee X8 machines (8 tips). They take about 10 minutes to set a 96-drop plate including the washes of the tips. 3 or 4 drops per condition wouldnt take much longer. Optimisation and additive/detergent screens take a little less time. The plates are pipetted under a close-fitting cover to (virtually) eliminate evaporation, which IMO is better than a humidity chamber. Consumable costs extend to isopropanol and water, with the occasional replacement valve or tip. Since people here also tend to turn up at beer o'clock on a Friday evening (must be an Oxford thing...) we have two machines (and another one imminent) to increase throughput. HTH Tom ** Tom Walter B.Sc. M.Res. ** ** Oxford Protein Production FacilityTel: +44 (0)1865 287747 ** ** Wellcome Trust Centre for Human Genetics Fax: +44 (0)1865 287547 ** ** Roosevelt Drive [EMAIL PROTECTED] ** ** Headington, Oxford OX3 7BNhttp://www.oppf.ox.ac.uk ** Original message Date: Mon, 14 Apr 2008 17:10:26 -0400 From: JOE CRYSTAL [EMAIL PROTECTED] Subject: Re: [ccp4bb] Fwd: [ccp4bb] crystallisation robot To: CCP4BB@JISCMAIL.AC.UK Hi, Does anyone have information about how long it takes to set up a 96-well tray for the crystallization robots available? Besides cost per tray and maintenance cost, another important feature we consider is the time for setting up a 96-well tray. It is an important factor since we are talking about sub-microliter drops. Best, Joe On Fri, Jan 18, 2008 at 12:28 PM, Lisa A Nagy [EMAIL PROTECTED] wrote: Al's Oil on the plates: What a nightmare!!! The oil creeps up the plate and over the sides. It dissolves adhesives. It makes me say bad words in multiple languages. Bigger drops + no oil = fewer bad words. Lisa -- Lisa A. Nagy, Ph.D. University of Alabama-Birmingham [EMAIL PROTECTED] -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Patrick Shaw Stewart Sent: Friday, January 18, 2008 2:20 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Fwd: [ccp4bb] crystallisation robot One thing that people often overlook is that quite a lot of protein can be lost by denaturation on the surface of the drop. This is more significant for smaller drops. Two suggestions: (1) increase the proportion of protein in the - technical term - teeny drop to say two thirds and (2) cover the drops with oil eg Al's oils (silicone/paraffin). You still get vapor diffusion though the oil , and you'd like to slow up equilibration. of course (2) slows up the robotics a little, but both should be trivial to set up..
Re: [ccp4bb] which concentrated salt has lowest vapour pressure?
This reminded me of a related article I came across a while back about controlling humidity with saturated solutions. # Saturated Solutions For the Control of Humidity in Biological Research # Paul W. Winston and Donald H. Bates # Ecology, Vol. 41, No. 1 (Jan., 1960), pp. 232-237 Hope that helps Tom ** Tom Walter B.Sc. M.Res. ** ** Oxford Protein Production FacilityTel: +44 (0)1865 287747 ** ** Wellcome Trust Centre for Human Genetics Fax: +44 (0)1865 287547 ** ** Roosevelt Drive [EMAIL PROTECTED] ** ** Headington, Oxford OX3 7BNhttp://www.oppf.ox.ac.uk ** Original message Date: Tue, 8 Apr 2008 13:42:21 +0200 From: Kay Diederichs [EMAIL PROTECTED] Subject: Re: [ccp4bb] which concentrated salt has lowest vapour pressure? To: CCP4BB@JISCMAIL.AC.UK Dear all, once again this bulletin board proved to be a great ressouce. I obtained 15 emails since I posted - thanks to everybody! I composed a summary for the CCP4 wiki, which can be found at: http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Crystallization_screens_and_methods#Desiccation_of_an_existing_screen_which_shows_no_sign_of_crystallization_or_precipitation thanks again, Kay Kay Diederichs schrieb: Dear all, a protein which we work on is available in low quantity. The only crystallization screen we set up is completely clear, no precipitate, nothing. Now we would like to modify the reservoirs of this screen, by adding LiCl or Ammoniumsulfate or ... , with the goal of reducing the vapour pressure, to at least get the protein concentration in the drop into the range where something happens. Does anyone have advice as to which salt we should add (to the reservoir only)? AmSO4 is only soluble to 4M, LiCl goes to 10M. But vapour pressure reduction is not the same as molarity. thanks for any insight, Kay -- Kay Diederichshttp://strucbio.biologie.uni-konstanz.de email: [EMAIL PROTECTED]Tel +49 7531 88 4049 Fax 3183 Fachbereich Biologie, Universität Konstanz, Box M647, D-78457 Konstanz smime.p7s (5k bytes)
Re: [ccp4bb] crystallization robot
Hi Madhavi In conjunction with a 96-syringe hydra for filling reservoirs, we have been using two Genomic Solutions 8-tip Cartesian systems (Called HoneybeeX8 now I believe) for the past 6 years and have been generally very happy with them. They require a bit of looking after in terms of cleaning, degassing and general maintenance (like most sensitive machines: have a responsible person), but have set up about 3 plates by now with 100nl + 100nl drops. We use it routinely for optimisation experiments and additive/detergent screens as well as initial screening. Consumable costs are pretty low (a few valves, tips and wash pumps over the years, along with a supply of isopropanol and water). The Innovadyne system is apparently quite similar but I have no experience of this. A few points to bear in mind though: 1) Evaporation of drops with small drop sizes - 100nl drops take only a few minutes to dry out completely, so you should either use a close-fitting cover over the plate, some kind of humidity chamber (each of the wells will be different though), have very fast plate setup time or chill the plate (which may affect protein solubility). We use a close fitting cover since each plate of 96 takes 10 minutes. 2) Contact and non-contact dispensing. Non-contact dispensing seems to be more accurate in terms of drop positioning in the well: important for images from automated imaging systems, image analysis software, and for fishing crystals out to test. Non-contact dispensing won't give you so many problems with low surface tension solutions such as detergents, and is compatible with hydrophobic plates (important for membrane proteins). I dont think there is a perfect robot out there - all the robots I have seen have both advantages and disadvantages so it just depends what you will be using them for, what disadvantages you are willing to put up with, and of course your budget. Happy shopping! Tom ** Tom Walter B.Sc. M.Res. ** ** Oxford Protein Production FacilityTel: +44 (0)1865 287747 ** ** Wellcome Trust Centre for Human Genetics Fax: +44 (0)1865 287547 ** ** Roosevelt Drive [EMAIL PROTECTED] ** ** Headington, Oxford OX3 7BNhttp://www.oppf.ox.ac.uk ** Original message Date: Wed, 9 Jan 2008 09:56:34 -0500 From: Nalam, Madhavi [EMAIL PROTECTED] Subject: [ccp4bb] crystallization robot To: CCP4BB@JISCMAIL.AC.UK Hi, Sorry for the non-ccp4 related question. We are planning to buy a crystallization robot. We looked at the 'Mosquito'. We felt it is good for setting 96 well plates (for screening the conditions). Though they say that we can use it for 24 well plates (hanging drop) it didn't seem to be ideal because all it does is set the drops and everything else has to be done manually. Can anyone suggest us other crystallization robots out in the market that are good? Thanks in advance, Regards, Madhavi
