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 <alun.co...@ucl.ac.uk
<mailto:alun.co...@ucl.ac.uk>> 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
<frank.vonde...@sgc.ox.ac.uk
<mailto:frank.vonde...@sgc.ox.ac.uk>> 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
<patr...@douglas.co.uk <mailto:patr...@douglas.co.uk>> 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
<519198...@163.com <mailto:519198...@163.com>> 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
--
patr...@douglas.co.uk <mailto:patr...@douglas.co.uk>
Douglas Instruments Ltd.
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University College London
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Web:www.ucl.ac.uk/pxmed <http://www.ucl.ac.uk/pxmed>
--
patr...@douglas.co.uk <mailto:patr...@douglas.co.uk> 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 <tel:01488%20649090> US toll-free
1-877-225-2034
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
