I assume you're sure you even *need* to scale up? Most of our
structures come from crystals from "small" (150-300nl) drops, we
consider a 100um crystal already "huge". And if a smaller crystal
doesn't diffract far enough on a modern beamline, chances are a large
one won't either (quite apart from the trouble you'll have
cryo-protecting it).
(And yes, of course there *are* a few cases where larger = better.)
phx
On 18/08/2010 19:39, Mo Wong wrote:
Thank you Patrick for your reply.
As a note to others who might be interested, I found a few comments
about scaling up interwoven in a long thread about which robot to buy
that was posted on this bb a few years ago. The most salient link is
probably:
http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg04387.html
Also, I found Patrick has a more detailed description about what
should be of primary consideration during scale-up written in the
following post:
http://groups.google.com/group/oryx_group/browse_thread/thread/b04a2d7736d5974d?pli=1
Regards
On Wed, Aug 18, 2010 at 12:56 PM, Patrick Shaw Stewart
<patr...@douglas.co.uk <mailto:patr...@douglas.co.uk>> wrote:
Hi Mo
What you need to remember is that a relatively large amount of
protein is lost from smaller drops. The ratio of surface area to
volume is greater. With 100 + 100 nl drops about half of the
protein is lost, either as skin on the drops or on the plastic of
the plate.
So when you scale up you need to reduce the protein by about
half. (Another approach, suggested by Heather Ringrose, is to put
extra protein into the drops at the screening stage, e.g. 200 nl
protein + 100 nl reservoir solution. The hits found can usually
be scaled up by dispensing 1 + 1 microlitre drops.)
This is counterintuitive because people expect the small drops to
dry out more quickly - so they expect, if anything, to get more
precipitation in the small drops. Instead they get precipitation
when they scale up, assuming they keep the ratio of protein to
reservoir constant.
It can also help, when you scale up, to increase the salt by 50 to
100% - this is indicated by data mining but I’m not sure what the
mechanism is
Hope that’s helpful
Patrick
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*From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK
<mailto:CCP4BB@JISCMAIL.AC.UK>] *On Behalf Of *Mo Wong
*Sent:* 18 August 2010 16:18
*To:* CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
*Subject:* [ccp4bb] Scaling up from an Intelliplate to Linbro Plate
Hi all,
I know scaling up from a hit found from a high throughput screen
is an empirical process, but does anyone have a good rule of thumb
as a starting point when it comes to scaling up from a hit
observed in an Intelliplate to a Linbro plate (i.e., change in
volume ratios, amount to add to reservoir, etc)? I've Googled
around but haven't seen anything which either suggests I shouldn't
be asking this question, I've not looked hard enough, or it really
is a case of "try and see".
Thanks
