methods for proteins
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|These generic diagrams show the
different areas of a protein-precipitant equilibrium in terms of the
concentrations of both components.
Areas shown in yellow (left) and white (right) represent the conditions under which the protein is in solution. The areas depicted in blue represent the conditions under which the protein appears as a precipitate. Both areas are separated by another area (shown in pink) with some supersaturation conditions, suitable for nucleation and crystal growth.
Thus, to maximize the possibility of a successful crystallization experiment, it is necessary to design various experiments, from different starting positions, ie different concentrations of protein and precipitant (arrows of different colours in the diagram on the right).
Scheme of a well reservoir, containing a precipitant solution, capped with a cover slip, as used in the hanging drop technique.
|The most common setup to grow protein
crystals is by the hanging
A few microliters (1-2 μl) of protein solution are mixed with a more or less equal amount of reservoir solution containing the precipitants (previously balanced with a pH buffer) and are deposited on a cover slip which covers the precipitant reservoir. As the protein/precipitant mixture in the drop is less concentrated than the reservoir solution (normally we mix the protein solution with the reservoir solution at about 1:1), water evaporates from the drop into the reservoir. As a result the concentration of both protein and precipitant in the drop slowly increases, and crystals may form.
The market offers several types of plates suitable for protein crystallization techniques, as it is shown below. See also the web pages offered by Hampton Research.
The interested reader can also have a look into this nice summary on the experimental aspects of protein crystallization. In case of problems use this link to watch the video offered by that website.
|Under the right conditions, crystals can grow in the drop as shown in the picture.||The available crystallization robots can help to prepare very quickly hundreds of drops to find the best initial conditions for crystallization.|
|The film on the left box shows the
process where lysozyme crystals are growing from an
The duration of the process, that takes a few seconds on your screen, corresponds approximately to 30 minutes in real life.
This case corresponds to an extremely quick crystal growth process.
The original film can be found on the website that offers George M. Sheldrick.
|With the same
enzyme (lysozyme), the
video produced by Bernhard Rupp shows the relationship between rapid
nucleation and crystal growth rate.
The greater the number of nuclei formed, the lower the growth rate, and therefore the smaller the size of the crystals obtained.
Compare the size (much smaller) of the crystals growing in the movie of the left with those shown above.