Tutorial 2: Precipitates

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" precipitate? Here are some things to look for (visual examples of these are given in the tutorial):

"good" precipitates

 "bad" precpitates

 non-amorphous precipitate

 characteristic brownish tinge to the precipitate

precipitate shows birefrigence

 skins on the drop

 will redissolve, if given the right conditions

 do not redissolve upon dilution

 

Remember: click on any picture to get a mangified view.

 

  A light, amorphous precipitate. 

 

 

 A heavy, amorphous precipitate. Most of the protein in the drop has precipitated.  

 

  This is NOT an amorphous precipitate. Look for things like this in your drop--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.

 

 Another example of an atypical (i.e. non-amorphous) precipitate in this microbatch drop. Notice that the precipitate has a pattern or different regions.

 

  A few days later, crystals appeared in the drop.

 

 Crystalline precipitate/microcrystals. The 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 focussing through the entire depth of the drop, these microcrystals were discovered.

 

Brown amorphous precipitate. BAD! This brown tinge is characteristic of denatured protein. Denature proteins never crystallize. Okay, 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. 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 affects the limit of diffraction.

 

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.

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