You optimize that variable as well in scale-up. That is the problem with nano-drop screening- sometimes it is near impossible to scale up the drops. You have to re-screen around the conditions (response surface methods can conserve experiments) because the kinetics change so much between nano and big drops.
The smaller they are, the harder it is to scale up, so we compromise between protein consumption and scalability. I think with most of these machines, you _can_ tweak it to dispense in the 20-50nl range accurately. To do that, you have to avoid using an air gap- either by drawing (and diluting) your protein against a column of water, or by using some other liquid (oil) between your protein and the dispensing fluid. Many times these tiny drops yield crystals much faster because they have more surface area to volume. But once you have them- well- that's where I've pulled out my hair. Scale-up is hard, and for delicate proteins (>1% crystalline hits), near impossible from the 20 nl drops. That's the main reason we use bigger drops. In the early stages of a project, there is often a great deal of batch to batch variability in the protein samples. Minimizing this, and getting consistent polydispersity measurements can go a long way toward making scale-ups easier. All this screening is for naught if you need to optimize using a second batch of protein which behaves differently from the first, or if your first/only batch is degraded by the time you have hits to optimize. If you are fortunate to be producing your own protein, you can personally make sure you are producing consistent batches. The worst thing you can do is screen on the hearts, then optimize/scaleup with the tails (of the protein peak in the purification). Personally, I think having a liquid handling robot is almost as important as a crystallization robot. Lisa P.S. Confidential to various sales people: 1. If you had visited us, you would have known we were going to buy another robot. 2. If you had visited us, you would have known I was in the group. 3. If a member of our group didn't contact you and request information for purchasing your fabulous robot based on your website/mailings/ads, then your marketing people (not us) have the problem. 4. Sole source justification. Features and Price. 20K is a significant difference, and we didn't need the extra features on your machine. Or we needed features yours didn't have. 5. Sending snarky email messages about me to my coworkers makes all of us less inclined to view your company favorably for future purchases. -- Lisa A. Nagy, Ph.D. University of Alabama-Birmingham [EMAIL PROTECTED] -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Oganesyan, Vaheh Sent: Wednesday, January 16, 2008 9:06 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] crystallisation robot ... Once in a while I get large crystal that can be used for data collection, but in most of the cases optimization in hanging 1+1 uL drops is required. What I found quite difficult to do is to choose the appropriate protein concentration when moving from 200+200nL to 1+1 uL. Sometimes protein should be diluted 3-4 times, sometimes it shouldn't. How others are approaching this issue? Vaheh
