Joe
The most common reason why people have difficulty scaling up is that they put too much protein in the drop. This may be true in your case because you report that you are getting heavier precipitation. What people don't realize is that you lose a lot of protein on the surface of the plastic and on the air/protein interface. Of course the amount lost varies from protein to protein, but in my experience and observation you typically lose half of the protein in 100 + 100 nl drops. So you have to decrease the amount of protein when you scale up (the surface area to volume ratio is lower in larger drops). I would have expected reducing the ratio of protein to well solution to have worked, but it's not quite the same thing as diluting the protein, especially if you're using PEG which doesn't give much equilibration. Try diluting the protein to say 30 - 60% of the original. On a different tack, I would instantly try MMS microseeding into random screens in a case like this. See D'Arcy et al. (2007). Acta Cryst. D63, 550-554 and Obmolova et al. (2010). Acta Cryst. D66, 927-933 Good luck 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 [email protected] Douglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, 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 From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Joe Sent: 23 November 2010 17:24 To: [email protected] Subject: [ccp4bb] Your suggestions needed: Difficulties in reproducing HT crystallization conditions. Thanks! Hi all, I recently have problems reproducing some conditions identified from high throughput screenings. The initial screening (10 mg/ml protein, 0.5 ul well+ 0.6 ul protein, 23 C) gave rise to at least three hits from different screen kits. The follow-up grid optimization (1.5 ul well + 1.5 ul protein) varying precipitant concentrations and pHs did not produce any crystals for all three conditions. Instead, the drops have heavier precipitation background. The following experiments have been done in order to get crystals back. 1. Seeding, with various precipitant concentrations 2. Varying volume ratios between well solution and protein (from 2: 1 to 1: 2 v/v). 3. Using original solutions from screen kits. 4. Hanging drops and sitting drops, 5. Dispensing protein first or well solutions first. 6. Using robot to set up drops on 96-well plate to see if I can reproduce the original hits. But none of them has worked so far. This is the first time I encountered such a scale-up issue. I am running out of ideas, so hope you could give me some suggestions. Thank you in advance. -- Best regards, Joe
