Yes, we have our screenmaking robot programmed to set 50% diluted screens and frequently employ this when a large fraction of the undiluted screens result in precipitation. We have found quite a few productive hits this way, as some previously grungy wells often present crystals when diluted.
Cheers, Roger Rowlett On Jul 12, 2017 12:01 PM, "Alun R Coker" <[email protected]> wrote: > So, if we have a commercial 96 well screen where more than around 60% of > the drops precipitate. It may be worth diluting the whole screen say (30ul > screen and 20ul water in each well) and repeating ..... rather than > diluting the protein. > > Has anyone ever tried this? > > All the best, > > Alun > > On 12/07/17 16:54, Frank von Delft wrote: > > Yes, exactly. Thanks for doing the Right Thing and posting the actual > diagram. > > > On 12/07/2017 16:26, Patrick Shaw Stewart wrote: > > > Alun > > I agree Frank's point is very interesting - and he intriguingly refers us > to the phase diagram. > > Is the point that Line A is longer than Line B ? > > Best wishes > > Patrick > > > > > > > > > > > > On 12 July 2017 at 14:40, Alun R Coker <[email protected]> wrote: > > Hi Everyone, > > Franks point is really interesting. We routinely reduce the protein > concentration when we see too many precipitated wells, but we never dilute > the screen. Has anyone tried this? > > All the best, > > Alun > > On 12/07/17 08:48, Frank von Delft wrote: > > The point I was failing to make: reducing either protein or precipitant > concentration will indeed reduce nucleation, but often won't get you bigger > or more single crystals: it will just make the appearance of crystals less > reliable. > > The way to get big single reliable crystals is to *increase* protein and > *greatly* reduce precipitant. > > (Even better: do seeding. Like Vicky said. Incredible how often people > don't bother to do seeding, yet it solves so many problems.) > > phx > > > On 12/07/2017 07:50, Vicky Tsirkone wrote: > > Dear Frank, > > I may see in the attached pic several nucleation points and a considerable > amount of microcrystals. Based to my knowledge decreasing the concentration > of the precipitant and/or the protein concentration would be a reasonable > approach to refine the initial hits. > By checking the diagram as you correctly mentioned you may see that the > fine tuning of protein and precipitant concetration may lead to the > desirable result without reaching the precipitation zone. > > Patrick just check your screens. Just a rule of thumb, if you see > precipitation in the ~60% of your drops then you should definitely reduce > the protein concentration. > > ps dont forget to try the *streak seeding*, as well. > > Have a nice day and again good luck. > > Vicky > > On Wed, Jul 12, 2017 at 8:50 AM, Frank von Delft < > [email protected]> wrote: > > Actually, you should try *increasing* the protein concentration - a lot. > But be prepared to drop the precipitant concentration to almost nothing (1 > or 2% isn't "low"). > > To understand why, look at the phase diagram and what we assume about > vapour diffusion. (Which I'm assuming is what you're doing.) > > > On 12/07/2017 06:28, Vicky Tsirkone wrote: > > Dear Patrick, > > You may reduce the protein concentation, as well. > Another option could be the *streak seeding* by exploiting the drop of > your initial condition. > > Good luck, > > V.T. > > On Mon, Jul 10, 2017 at 7:17 PM, Patrick Shaw Stewart < > [email protected]> wrote: > > > Microseed them into two or three random screens. > > Search for MMS and rMMS online. > > Good luck > > Patrick > > > > > On 10 July 2017 at 15:47, Liuqing Chen <[email protected]> wrote: > > hello everyone! > I get a condition (10% w/v PEG 6000, 100mm HEPES PH7.0) in which my > protein grow small needle like crystals, how can i optimize it to get > bigger crystals? the attach is the crystals figure. > thanks in advance > sincerely > Liuqing Chen > > > > > -- > [email protected] Douglas 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) 148-864-9090 <01488%20649090> US toll-free 1-877-225-2034 > <%28877%29%20225-2034> > Regd. England 2177994, VAT Reg. GB 480 7371 36 > > > > > > > -- > Dr Alun R. Coker > Senior Lecturer > Wolfson Institute for Biomedical Research > University College London > The Cruciform Building > London > WC1E 6BT > > Tel: 020 7679 6703 Ext 46703 <020%207679%206703> > Web: www.ucl.ac.uk/pxmed > > > > > -- > [email protected] Douglas 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) 148-864-9090 <01488%20649090> US toll-free 1-877-225-2034 > <(877)%20225-2034> > Regd. England 2177994, VAT Reg. GB 480 7371 36 > > > > -- > Dr Alun R. Coker > Senior Lecturer > Wolfson Institute for Biomedical Research > University College London > The Cruciform Building > London > WC1E 6BT > > Tel: 020 7679 6703 Ext 46703 > Web: www.ucl.ac.uk/pxmed > >
