One of the most difficult things for beginners is to recognize promising precipitates and distinguish them from preciptiates that are not worth pursuing. Except for drops where the protein is denatured, precipitate can be regarded as a positive drop result. This is because precipitate occurs in a state of supersaturation. Crystals can only grow from supersaturated states, and thus they can grow from precipitates.
How do I tell a good precipitate from a bad one? (See the pictures below for examples.)
|Non-amorphous precipitate||Characteristic brownish tinge to the precipitate|
Precipitate shows birefringence or is shiny white
|Skins on the drop|
|Precipitate redissolves if given the right conditions||Precipitate does not redissolve upon dilution|
Precipitate or nanocrystals?
Many so-called precipitates are actually nanocrystals, but this is not possible to detect with optical microscopy. These nanocrystals (if they are chiral) are identifiable by another technique, SONICC (second-order nonlinear imaging of chiral crystals). In a study on 2000 drops, nanocrystals were identified by SONICC in over 90% of the precipitates.
Although most laboratories do not have access to a SONICC, there is a take-home message here that can be applied in any laboratory. As many precipitates are, in fact, crystalline, use the precipitates as seeds. If you have nothing better in your screening results than precipitates, try seeding with them.
Of course, you can try manipulating the protein and precipitant concentrations. But if this does not help, use the precipitates as seeds and do matrix microseeding. (See the seeding tutorial.)
A light, amorphous precipitate
A heavy, amorphous precipitateMost of the protein in the drop has precipitated.
Non-amorphous precipitate 1
This is NOT an amorphous precipitate. Look for things like this in your drop, i.e., precipitates which have some kind of anomalous pattern. Do you notice the darker, localized regions? These are often pre-cursors to crystals or at least spherulites.
Non-amorphous precipitate 2Another example of an atypical (i.e. non-amorphous) precipitate in this microbatch drop. Notice that the precipitate has a pattern or different regions.
Non-amorphous precipitate 2A few days later, crystals appeared in the drop.
Crystalline precipitateThe student who showed me this drop completely missed the crystals because: 1. he had only focused at one depth in the drop, and 2. only checked the drop at 40x magnification. Notice the blurry region in the upper one-third of this picture. If this region is IN focus, then the tiny microcrystals underneath it are OUT of focus and cannot be seen. By changing to 100X magnification and focusing through the entire depth of the drop, these microcrystals were discovered.
Crystalline precipitate: whitish color against a black background
more info coming soon
Brown amorphous precipitate. BAD! This brown tinge is characteristic of denatured protein. Denature proteins never crystallize. You might yet get crystals in this drop, but only if enough of the un-denatured protein is still in a state of supersaturation. This is unlikely, but still possible. Note the skin on the drop; see below.
Skin on the drop. BAD! These skins are believed to be a layer of denatured protein or caused by the polyethylene glycol. Avoid them, they will certainly slow down the rate of vapor diffusion. Pick away the skin before mounting your crystal. If the skin wraps around the crystal it gives a heavy background in your diffraction pattern.
Precipitate with crystal
Crystal growing from a heavy, amorphous precipitate.
It often happens that precipitate occurs first and later crystals grow. For more examples of this see tutorial 6 on Ostwald ripening.