[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
Re: [ccp4bb] Scaling up from an Intelliplate to Linbro Plate
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 -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Mo Wong Sent: 18 August 2010 16:18 To: 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
Re: [ccp4bb] Scaling up from an Intelliplate to Linbro Plate
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 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 -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of *Mo Wong *Sent:* 18 August 2010 16:18 *To:* 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
Re: [ccp4bb] Scaling up from an Intelliplate to Linbro Plate
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 -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en patr...@douglas.co.uk mailto:patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 *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
