Waiting the protein to concentrate: setting up your crystallization experiments for success
Posted by Peter Nollert on Tue, Oct 27, 2009 @ 12:43 PM
Protein samples need to be concentrated prior to setting them up in a protein crystallization experiment, ideally to above 10 mg/ml. Since this concentration business takes a lot of patience, it's a good idea to plan ahead for a successful protein crystallization trial:
• Know what your target concentration is in OD units rather than in mg/ml - no need to waste any time with Beer-Lambert.
• Workplace: bring out all your tools: crystallization plates, sample container, pipettors, tips, pens, stands, notebook, box for used tips, Wizard screening kits ;). Arrange everything on your bench so you're ready 'to do the robot'. Label your crystallization trays (not the lid).
• Prepare for a steady hand and some quiet time. I used to enjoy setting up crystallization trays all by myself, coffee abstinent for 4h (otherwise I'd get the jitters...). Less traffic in the lab means fewer distractions, helping to avoid pipetting errors.
• Using Crystallization Robots? It almost goes without saying that at this point you want to have had a training session in proper use of the machine. Do clean any liquid carrying pathways, check the waste containers and all the lines. Get all the accessories in place, you don't want to run out of those special pipetting tips or clog a needle. Running a single trial dispense doesn't hurt either.
• Prepare to shock-freeze in liquid nitrogen a sample of the concentrated protein, round up the dewar with liquid N2, safety goggles, vials and tongs. This is for the positive control and follow-up optimization trials. I used to work in a lab where getting liq. N2 was a pain, involving taking the elevator into the basement and then operating a scary piece of equipment making loud unpredictable noise. I learned the hard way that N2 levels were usually low towards the end of the week.
Now you're getting close to combine your perfectly formulated protein solution with sets of crystallization reagents.
Here's the pre-crystallization setup countdown:
t= -15 min: Centrifuge stops, sample out
t= -10 min: OD280 is within your target range.
t= -5 min: Shock freeze the portion of protein that you're not planning to set up. Freeze the solution you're not using with this crystallization trial later
t= 0 min: pipette!
While it's not rocket science, proper preparation for the crystallization experiment minimizes errors and sets you up for a successful protein crystallization trial.
No more "Huston, we have a problem".
Peter
P.S. Somewhat related to this post: Michael Sawaya has written up a nice intro on "What every crystallographer should know about a protein before beginning crystallization trials"
