Joe- You might want to try your original drop ratio of 0.6 ul protein + 0.5 ul well in your optimizations in the 96- well format used to obtain your initial crystals.
HTH! annie From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Joe Sent: Tuesday, November 23, 2010 12:03 PM To: CCP4BB@JISCMAIL.AC.UK 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
