Hi Joe, I really want to re-iterate both Annie and Patrick's suggestion of trying different drop ratios and protein amounts, however you might also try adding some fresh reducing agent, particularly if you are trying the scale-up with protein that has been hanging around (or sitting around) since the screening.
Regards, Janet Dr Janet Newman Principal Scientist | C3 Facility Manager Materials Science and Engineering CSIRO Phone: +61 3 9662 7326 | Fax: +61 3 9662 7101 | janet.new...@csiro.au | www.csiro.au | www.csiro.au/c3 Address: 343 Royal Parade, Parkville VIC 3052 AUSTRALIA PLEASE NOTE The information contained in this email may be confidential or privileged. Any unauthorised use or disclosure is prohibited. If you have received this email in error, please delete it immediately and notify the sender by return email. Thank you. To the extent permitted by law, CSIRO does not represent, warrant and/or guarantee that the integrity of this communication has been maintained or that the communication is free of errors, virus, interception or interference. Please consider the environment before printing this email. From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Joe Sent: Wednesday, 24 November 2010 4:03 AM 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
