Spherulites, gels, phase separations…

Spherulites

Spherulites can be difficult to distinguish from oils unless you probe them with a whisker or needle. (Oils are dense liquid phases, kind of like honey; spherulites will be crunchy, solid.) Another feature that differentiates them: look closely at the edges of the spherulites. They are not completely round, as would be a liquid-liquid phase (oil).

What is the difference?

If you can't see the difference between an oil and a spherulite, don't worry. In practice, it doesn't really make any difference: treat the oils or spherulites in the same manner. Use them as seeds in a new drop. Of course you can also screen around the conditions. Maybe all that is required is fine-tuning the concentrations of precipitant, etc.

Gel

Synonyms: Gelationous protein, gelled protein

This is my favorite example of a gel because the protein is red (it is a heme-binding protein). When this gel materials was used to streak-seed new drops, good crystals grew.

Gel

Transparent, irregular regions in the drop. You are very close to the right conditions. In this example, the large crystal in the center of the drop grew some weeks after the gel. Some of the gel is covering the right side of the crystal.

Gel

Another example of what gels look like, this time in a microbatch drop. Compare the difference between gels and amorphous precipitates. Gels have at least some kind of short-range order to them. Do a grid screen or an additive screen around the condition where you obtained the gel.

Gelled protein

Another example of gelled protein, this time a close-up view. When you see this in your drops, you are very close to the right conditions; screen around the conditions that give you this.

Liquid-liquid phase separation (LLPS) or oils

A classic example of a liquid-liquid phase separation is sallad dressing (oil & vinegar). Shake it. What does it look like?

LLPS in the crystallization drop will often appear as hundreds of small droplets. If a lot of the small droplets coalesce (merge together), you will get a great big drop, like the one you see here in the middle.

The protein will preferentially "oil out" into one of the liquid phases more than the other. (You can only have two phases simultaneously.) When the protein "oils out", it forms a very dense liquid. Hence, liquid-liquid phase separation and "oils" are two descriptors of the same phenomenon.

Click to learn more about phase separation and get tips on how to optimize it. 

Liquid-liquid phase separation with a crystal

In this example, a huge crystal (>0.3 mm in the longest direction) grew some weeks after the  phase separation. So, yes, you can get crystals in drops with phase separation, just as you can get crystals growing in drops full of precipitate. 

Crystals growing in LLPS

Here we clearly see that the protein has preferentially sequestered itself into one of the phases. It becomes highly concentrated in the phase, and the high levels of supersaturation in this phase are more conducive to nucleation than in the other phase. These crystals show up beautifully in the UV imaging (see next image).

UV image with LLPS

What to do next though? Obviously these crystals are too small to mount, so why not use them as seeds.