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 <[log in to unmask]> 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:[log in to unmask]] On Behalf Of
> [log in to unmask]
> Sent: Wednesday, January 16, 2008 8:31 PM
> To: [log in to unmask]
> 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 <[log in to unmask]>
> To: [log in to unmask]
> 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: [log in to unmask]
> > URL: http://athena.eie.gr
> > ==================================================
>
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