In an ideal world, this would be a fairly simple way to grow protein crystals for X-ray structure determination purposes:
Start out with ca. 400 ul of filtered target protein solution, in 20 mM Hepes buffer neutral pH and maybe 50 mM NaCl , freshly purified to ca. 95% purity and concentrated to 10 mg/ml.
1. Snap freeze four x 25 ul aliquots of protein samples in thin-walled PCR tubes and store at -80C. These sample will be used in a week for protein crystallization optimization.
2. Document the quality of the protein sample via SDS-PAGE and determine its concentration (ca. 5 ul). Measuring UV absorption (OD280) and calculating the protein concentration is good enough. You'll need ca. 5 ul for such an OD measurement (depending on cuvette path length and dilution). Check this online tool to get an estimate for the extinction coefficient based on sequence.
3. Now let's get out the multichannel pipettor and set up all of the remaining 200 ul of sample solution as 1ul + 1 ul sitting drop vapor diffusion crystallization experiments. A great first pass would look like this:
Plate 1: Wizard 1 & 2 (96 drops)
Plate 2: Wizard 3 & 4 (96 drops)
By the way, Emerald offers these reagents as the Wizard Suite. This is a 192 point, non-overlapping, sparse matrix that has a proven track record to yield crystals in first-pass protein crystallization exploration trials.
And while we're at it: go with the Compact Jr. plates. These plates can be sealed with clear tape and drops form nicely on the hyrdophobic polypropylene surfaces.
4. Store the crystallization trial at room temperature, minimizing temperature fluctuations and vibration. Observe right away, after 1 day, 3 days and 1 week.
5. The initially clear drops with now contain precipitate, some clear drops and a few with clustered microcrystals or needles in them. If the corresponding well-solution does not contain similar crystals, chances are that you've grown protein crystals! Since these crystals are likely to be too small to diffract them using your home-source X-ray generator and detector system, you'll need to optimize crystallization conditions and grow larger crystals.
6. There are many different ways to optimize crystallization conditons, and depending on prior knowledge you may want to carry out seeding experiments, include additives or change the treatment of the protein sample (i.e. filtration). Here's a simple optimization schema: create a grid screen around all conditions that gave you crystals. This rational crystallization optimization schema works great since it separates the effect of pH, salt, precipitant and protein concentration). You should use the online ScreenBuilder design tool to create an optimization screen. Your fellow researchers at Emerald BioSystems are happy to prepare and send such a customized optimization screen to you.
7. Set up 96 - follow-up optimization 1 ul + 1 ul crystallization experiments with the saved protein material that you thaw in your fingers. If everything goes according to plan, crystals of different sizes will grow.
8. Harvest a crystal, cryoprotect, diffract and determine its structure ;)