Hi Matt Rajesh asked a similar question last week, below
Essentially, you have to *reduce *the protein concentration when you scale up because you lose proportionally more protein from smaller drops. This usually works very well and we see no reason to use more than 0.3 + 0.3 for initial screening. There's a page on our web site which explains this in much more detail, see http://www.douglas.co.uk/Scaling_Up.htm Best wishes Patrick ---------- Forwarded message ---------- From: Patrick Shaw Stewart <patr...@douglas.co.uk> Date: 19 March 2012 22:29 Subject: Re: [ccp4bb] microseeding To: Rajesh kumar <ccp4...@hotmail.com>, ccp4bb@jiscmail.ac.uk Rajesh If you set up the volumes you suggest you will probably get precipitation. This is counterintuitive until you realize that (as Ed says) you will be losing a lot of protein with those small drops. When you scale up the surface area to volume ratio is lower, so a smaller proportion of the protein is lost. Therefore you go *up* on the phase diagram and get precipitation or very small crystals. Normally halving the amount of protein for the hits from 200 nl drops works (suggesting that half the protein is lost from such small drops). Try say 500+1000+500 (don't reduce the volume of seed stock because the solution that you suspended the crystals in may be important). Or dilute the protein and use 1000+1000+500. For the hits from the 450 nl drops you could reduce or dilute the protein by say 25.%. Or make plenty of seed-stock and try seeding into a random screen again with larger drops, say 1.5+1+0.5 ul Those tiny crystals should be good for seeding, don't worry about that (provided they are protein of course). Streak seeding may work but bear in mind that roughly a third of the precipitant comes from the seed stock in your 250 nl drops. You can add the seed stock with a syringe and needle if you don't have suitable robot ;) Experience and data-mining suggests that increasing the salt precipitant (in high-salt drops) or salt additive (in PEG drops) by around 50% may be helpful too when scaling up - I'm not sure why this works. Good luck Patrick For the hits in the 250 nl drops you are probably losing On 19 March 2012 20:31, Rajesh kumar <ccp4...@hotmail.com> wrote: > Dear All, > > I have few papers in hand which explain me about microseeding, matrix > microseeding, and cross seeding. > I have also read few earlier threads and some more literature in google. > Using Phoenix robot, I did a matrix micro-seeding and matrix cross > seeding. I have few hits with this. > In 96 well I used 100+100+50 nL and 200+200+50 nl (protein+screen+seed) in > separate expts. > I have hard time to plan to translate this 96 sitting drop well plate to > 24 well plate to refine the conditions to get better crystals. only 1-2 > hits are small crystals and they are tiny. > > I wonder in 24 well plate, if I should do- > 1) for Example 500+500+50nl (I am sure I cant add less that 500 > nL precisely) > 2) to a drop of 500+500 nL do microseeding/streaking with a hair > > I appreciate if you could advise and share some practical ways to further > my experiment. > > Thanks in advance > Regards, > Rajesh > -- 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 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 -- 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 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
