Protein Crystallization Hits

Let's say you have plenty of protein sample available and you want to grow really large crystals. How large? No-microscope-necessary-to-see-them large. Here's how to do it:

Small-scale batch technique

Ivan Rayment describes in his paper

Rayment I. 2002. Small-scale batch crystallization of proteins revisited: An underutilized way to grow large protein crystals. Structure 10: 147-151

that large protein crystals can be grown (for ca. 70% of proteins) in this way:

1. Forget vapor diffusion (no sitting drop, no hanging drop)

2. Know your initial crystallization condition (this method is not about screening for a crystallization hit)

3. Learn the slow mixing technique and prepare setups as small-scale batches

4. Identify the seeding sweet-spot (sub-nucleation conditions) 

5. Reproduce by scaling up in larger volume 

At first you'll need to 'translate' a given crystal growth condition to small batch crystallization. This is done by slowly mixing 60-80% of the precipitant with an equal volume of protein sample and placing it into a dimple glass plate with a seal. With a gradual variation of the precipitant concentration you'll determine the conditions that produce a supersaturated solution but no productive nuclei. The key is slow, homogeous mixing that Ivan describes like this: start with 5 ul of protein in the bottom of an Eppendorf vial, then vortex slowly (just 5-10 rotations per second) and add the precipitant solution s-l-o-w-l-y. For example over a period of 2 to 5 seconds. Don't mix by aspiration! The goal is to get into a homogenous mixing regime. The mixture is then transferred to the sealed container.
In the next step you want to identify the solution that is supersaturated but has not produced nuclei - a clear drop that produces crystals when seeded. This can be tested by streak seeding. Practically this is done by touching a crystal with a clean probe and streaking it through a clear drop. The drop that grows crystals after this procedure is suitable for the next step:

Scale up.

At this point you know how to produce a supersaturated solution of your protein and how to induce crystallization. Adjustments of precipitation concentration, pH, salt, temperature etc (sic!) may be necessary, but ideally you would just repeat the experiment with larger volumes, let's say 100 uL. If you can, use a test tube as the vessel to slowly add precipitant to the protein solution. On the vortexer. V-e-r-y   s-l-o-w-l-y. 

Taken from Ivan Rayment's paper 

All the best,

Peter