Drops.
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The drops are first checked at low magnification (40x), then more closely at l00x. Do not use polarized light, because of interference from the plastic tray with the polarized beam. At this stage the color of the precipitate is important.The results of the crystallization experiments are recorded using the numbering scheme. The score is used to classify the contents of the drop with one or more numbers, to save space in the lab book and for easy identification.
Example: A score of 49 = gelatinous precipitate (score 4) with crystals (score 9).
The scoring is not a quality scoring; a higher number does not necessarily mean a better result.
When screening for crystallisation condition, examine the conditions
The rate of formation is important for optimization of the precipitant concentration.
- immediately after setup
- each day for the first week
- once a week for several weeks. (store upto 2 years)
In the final stages of optimization, when we are aiming for highly ordered, big crystals, it is better not to disturb the crystal growth. Wait one or two weeks before checking the experiment.
The protein is not precipitated.
0. The drop is clear.
1. Drop contains non-protein particles.
Precipitate shows no birefringence and has no edges.
2. Drop is mostly clear but contains precipitated protein
3. Protein fully precipitated
4. Gelatinous or particulated precipitate
- The protein is fully precipitated.
- The precipitate looks white/transparent.
5. Phase separation
- Visible as small droplets in the drop, or as a film.
Precipitate shows birefringence or has edges.
6. Spherulites or microcrystals
- Transparent clusters, sometimes birefringent with visible needles.
- Small structures, where you might see the edges of the crystals
7. Needles
- Birefringent crystals but only grown in one dimension
8. Plates
- Very thin birefringent plates, not good enough for X-ray diffraction.
9. Crystals
- Do I have to explain?
© 12 Febr. 2004 Johan Zeelen