Protein Crystallization Hits

There's plenty of time to get all of your primary protein crystallization experiments prepared within a single day, including preparation for follow-up optimization experiments. Here's how you may want to schedule your protein crystallization day:

1. First thing in the morning:  get the protein out of the dialysis bag (or cassette) and transfer into the concentrator device of your choice. Save a small sample to determine the concentration:
2. While the sample is concentrating measure its light absorption at 280nm, using the dialysis buffer as a blank.  No need to run a calibration curve if you just shoot for 20 OD, i.e. concentrate your protein until the absorption at 280 nm has reached a total of 20 OD units. See here for how to go about this and what's behind this rule-of thumb.  
3. Once concentrated to 20 OD, quickly filter your protein sample with a filter, for instance a 0.1 micron filter to clean up the sample.

Depending on when you start your day in the lab, you will have the protein sample ready to go before noon or early in the afternoon. Now you've only got an hour or so to prepare the entire crystallization trial:

 4.  Setting up a single tray of sitting drop/vapour diffusion crystallizations takes less than 20 min, four trays should take less than one hour. Definitely include a sparse matrix screen such as the two tried-and-proven 96-well Wizard screens (Wizard I&II and Wizard III&IV).  

5. If you've got 300 uL of protein sample volume, first use ca. 160 uL to prepare 4 trays and  snap freeze the remaining volume in four aliquots without adding any cryo protectant once you're done setting up the crystallization trial (check out the best-practice snap freezing procedure here). Those aliquots will be useful for optimization of any hits or for reproducing the crystals that you get in this first screening pass.

6. Take a quick look at the first tray right after you have set it up and check if there's quick precipitation in many of the wells. If more than 1/2 of all the wells are cloudy, you may want to reduce the protein concentration by 1/3 or 1/2 for the remainder of the crystallization trial.

7. Note everything down  what you did, especially any screw-ups and unusual observations. These notes can help you in strategizing further crystallization attempts.

At this time it's time of a cup of tea.

Have a good crystallization day,

Peter