Not with membrane proteins, however we use our Mosquito with 20 nl seeding standard.
Strip 1 Protein, Strip 2 Seed stock solution We first copy the plate by either picking up protein, then reservoir and dispensing together 200-400 nl or we multidispense protein and add the reservoir later (larger drops, in particular if we can't concentrate the protein well then we add 600 nl and hope that enough evaporates before we come with the reservoir solution). As last step we add the 20 nl seed solution. We've also done seed titrations then we use three strips 1)Protein 2) seed buffer solution 3) seed. In that case the combination of 2+3 is always 100nl and we start out with 5 nl Seed stock. In the IntelliPlates (3well) we play the protein/reservoir ratio game + seed Reproducibility is pretty good. We keep our seed stock where we grew the crystals e.g. 20˚C or 4˚C. Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Sep 29, 2010, at 5:23 AM, Patrick Shaw Stewart wrote: > Hi Jeroen > > I think that part of the point of the method is that it’s incredibly quick > and easy to do – you can make seeds and run an experiment in 20 mins. But > I’m sure it’s important to use the seed stock quickly and freeze it. > > > Maria > > I think most people use 100 nl seed stock, 200 nl reservoir, 300 nl protein > as D’Arcy and co originally suggested. Maybe more seed stock would be better > as Roberto says, to increase the additive effect. The main reasons not to > put seed stock into the reservoir are (1) it’s more work – compared to your > robot picking up the seed stock from a PCR tube - and (2) sometimes your seed > stock is very valuable, for example if you only have one crystal and you make > up say 5 ul of seed stock. > > Has anyone had success with MMS microseeding with membrane proteins? I > wonder if membrane protein crystals dissolve more easily. > > Patrick > > > > -- > For information and discussion about protein crystallization and automation, > please join > our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en > > [email protected] Douglas Instruments Ltd. > DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK > Directors: Peter Baldock, Patrick Shaw Stewart > http://www.douglas.co.uk/ > Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 > Regd. England 2177994, VAT Reg. GB 480 7371 36 > > From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of mesters > Sent: 28 September 2010 07:26 > To: [email protected] > Subject: Re: [ccp4bb] Micromatrix seeding using the mosquito > > Hello Maria, > > the success of this method depends on a few things. > First, your seed stock-solution. Most people prepare it by smashing up seeds > in mother liquor (i.e. reservoir solution). Looking at the phase diagram, you > will quickly understand that this might not be the best way to go as you will > dissolve your seeds fairly quickly over time. That is why people prepare > really fresh stocks, keep them on ice and freeze them as soon as possible in > order to "stabilize" the seeds. Keeping on ice will only help if your protein > is less soluble at lower temperatures, which true for roughly 45% of proteins > (45% will react retrograde, 10% do not show a clear preference). Looking at > the phase diagram again, you will also understand that seeding only works for > "near" equilibrated drops that are metastable-supersaturated. If > undersaturated, seeding will not work as the seeds will dissolve over time. > > Keeping the above considerations in mind, best is to seed after the drops > have equilibrated. Next best option is to add the seeds or the protein as the > last component to the drop... > > Jeroen. > > > > > On 20.09.10 09:53, Maria Håkansson wrote: > Dear all, > > Anyone who have had success using the mosquito for micromatrix seeding? > Which way is the optimal way of adding seed solution to sitting drops on an > MRC plate? > > Is it better to add 100 nl protein + 10 nl seed solution + 100 nl reservoir > solution or > to add concentrated seed solution (1 microliter) directly to the reservoir > solution and then pipett > 100 nl protein + 100 nl reservoir solution. > > Best regards and thanks in advance, > > Maria Håkansson > > > > __________________________________________ > Maria Håkansson, Ph.D. > Senior Scientist, Max-lab, Lund University > Phone: +46 (0) 76 8585 706 > Fax: +46 (0) 46 222 47 10 > Ole Römers väg 1 (P.O. Box 188) > SE-221 00 Lund, Sweden > > Web address: www.maxlab.lu.se > Email: [email protected] > __________________________________________ > > > > > > > -- > > <image002.jpg>-- > Dr. Jeroen R. Mesters > > Gruppenleiter Strukturelle Neurobiologie und Kristallogenese > Institut für Biochemie > Universität zu Lübeck > Zentrum für Medizinische Struktur- und Zellbiologie > Ratzeburger Allee 160, D-23538 Lübeck > Tel: +49-451-5004065 > Fax: +49-451-5004068 > Http://www.biochem.uni-luebeck.de > Http://www.iobcr.org > Http://www.selfish-brain.org > Http://www.opticryst.org > -- > If you can look into the seeds of time and say which grain will grow and > which will not - speak then to me (Macbeth) > --
