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..
On 1/17/08, Oganesyan, Vaheh <[EMAIL PROTECTED]> wrote: > > > > > Mark, > > > > What was the state of the larger drops when tiny counterparts had crystals? > My guess - they all precipitated. > > I'm trying to understand why some proteins or some conditions require change > in protein concentration while others do not when migrating from smaller > drops to larger ones. If it is protein dependent then I'm afraid there might > be no one answer; if it is not then there should be a trend and explanation > of phenomena. > > > > > > > Vaheh > > ________________________________ > > > From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of > [EMAIL PROTECTED] > Sent: Wednesday, January 16, 2008 8:31 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] crystallisation robot > > > > > > Once upon a time I worked in a group that was interested in developing > crystallization in microfluidics. This was before the time that Fluidigm > existed and we had not heard of crystallization with the aid of > microfluidics at the time. We had good reason to try to make a system that > was as small and light as possible - it had something to do with the cost of > shipping proteins and precipitants - less was better. And we also wanted all > protein drops to be fully enclosed, out of safety considerations. > > Like Tassos, we were very worried what would happen if you scaled back > drops along the lines of this discussion - several uL downto tens of > nanoliters. If the stochastic process had a major influence over this > process, we thought that we would never get any crystals. So we set up > side-by-side experiments at larger volumes and smaller volumes - basically > scanning several orders of magnitude - expecting a decrease of the number of > crystals when volumes decrease. To our great surprise the outcome was that > smaller volumes almost always gave MORE (I almost want to say 'dramatically > more') crystals, more nucleation, and indeed in various cases the crystals > grew much faster also. Indeed, it was trivial to observe that the > surface-to-volume ratio was the primary driver for the nucleation process. > We had control over geometry to some extent and were able to observe > surfaces while crystals grow. The crystals would most commonly nucleate on a > surface. > > So although there probably is something to stochastic aspects, it is clear > that other aspects can be more important and "overrule" the stochastic > considerations. > The somewhat unpleasant consquence is of course that results acquired in > very small volumes (with larger surface-to-volume ratio) cannot necessarily > be repeated in larger volumes (smaller surface-to-volume ratio). > > This is not a flame, even if heat might be a good thing on a night with > temperatures predicted far below 0F. > > :-) > > Mark > > > > > > > > -----Original Message----- > From: Anastassis Perrakis <[EMAIL PROTECTED]> > To: CCP4BB@JISCMAIL.AC.UK > Sent: Wed, 16 Jan 2008 6:17 am > Subject: Re: [ccp4bb] crystallisation robot > > > > Oryxnano 50+50 nL > > > > Demetres > > > > Which, indirectly, brings up an interesting (but not relevant to the Oryx) > question. > > Nucleation is a process that does have a stochastic aspect. > > Thus, one could argue that compromising to 200-300 nl might be better than > either extremes of 50nl (too small volume and less chance for nucleation) or > 1000 nl (too much sample). > > any comments ? (let the flames begin). > > A. > > PS1 > another interesting issue that has has been hardly touched in these emails > is the real sample loss: left in wells and not easy to recover, lost because > of contamination with system liquid, etc ... > > PS2 > I see lots of people with new robots. please do have a look at the > www.BIOXHIT.org page and if you have a few minutes to assemble a table we > will be happy to add your specs to our pages. it can be a nice resource and > it has already enough things and already one response to my last email ;-) > To make life easier to potential contributors we can provide an Excel sheet > to fill up with your specs - just ask. > > On Jan 16, 2008, at 12:46, Demetres D. Leonidas wrote: > > > > > David Briggs wrote: > >> I'll defend the honour of the phoenix... (again) > >> > >> Bernhard Rupp 100+100 nl > >> Dave Briggs (and all users at Univ of Manchester, UK) 100+100nl > >> Others.. > >> > >> Only time we have ANY problems is when the nano dispensing tip >> gets > clogged. Often a good wash whilst still on the machine will >> clear the > blockage. > >> > >> Dave > >> > >> > >> > >> > >> -->> ============================ > >> David C. Briggs PhD > >> Father & Crystallographer > >> http://www.dbriggs.talktalk.net <http://www.dbriggs.talktalk.net> > >> AIM ID: dbassophile > >> ============================ > > > > --> Demetres D. Leonidas, Ph.D. > > Structural Biology & Chemistry Group > > Institute of Organic and Pharmaceutical Chemistry > > The National Hellenic Research Foundation > > 48, Vassileos Constantinou Avenue > > Athens 116 35, Greece > > ================================================== > > Tel. +30 210 7273841 (office) > > +30 210 7273895 (lab) Fax. +30 210 7273831 > > E-mail: [EMAIL PROTECTED] > > URL: http://athena.eie.gr > > ================================================== > > size=2 width="100%" align=center> > > More new features than ever. Check out the new AIM(R) Mail!
