Dear CCP4ers, Thanks a lot for all your replies. Additionally, I have to thank Art Robbins, Dave Wright and colleagues from Art Robbins Instruments for almost immediate replies and for taking care of our problems. Besides of the normal wash steps that we do and of course a well prepared protein sample (centrifuged at high speed, take supernatant), CCP4 users have suggested the following:
- Lots of Phoenix users do a 0.1M NaOH wash after each cycle (e.g. using reservoir 2 bottle), which seems quite reasonable and we have also introduced it on our system now. - More extensive wash steps in between the different users (longer NaOH or Zymit wash). Other users increased the frequency of Zymit wash-steps (1 zymit wash every 10-15plates). We are now using an end-of-day-wash protocol including Zymit and eventually do a Zymit wash every couple of users. - Prevent dust everywhere if possible (we use HPLC grade water for the system liquids in Reservoir 1 and 2, cleaned the bench etc.) - ARI suggested pre-wetting of the nozzles with sample buffer prior to aspirating the protein solution. This might prevent that "difficult proteins" feel even more unhappy if they touch dry surfaces or wet surface without any ions (like present after washing with water). I am going to try this. - As some of our proteins may indeed be difficult, ARI suggested a short 2M urea aspirate/dispense step after dispensing the protein, prior to the NaOH wash step. We have changed all nozzles to new ones and introduced the steps described above and at the moment it seems that we are running fine. Thanks again for all your help and merry Christmas to all of you out there! Gregor --- Dr. Gregor Witte Research Associate Genecenter, University of Munich (LMU) Feodor-Lynen-Str. 25 D-81377 Munich mail: [email protected]
