Methods of Crystallization
Methods of Crystallization
There are many methods to crystallize biological macromolecules (for reviews see refs 1-3), all of which aim at bringing the solution of macromolecules to a supersaturation state (see Chapters 10 and 11). Although vapour phase equilibrium and dialysis techniques are the two most favoured by crystallographers and biochemists, batch and interface diffusion methods will also be described. Many chemical and physical parameters influence nucleation and crystal growth of macromolecules (see Chapter 1, Table 1). Nucleation and crystal growth will in addition be affected by the method used. Thus it may be wise to try different methods, keeping in mind that protocols should be adapted (see Chapter 1). As solubility is dependent on temperature (it could increase or decrease depending on the protein), it is strongly recommended to work at constant temperature (unless temperature variation is part of the experiment), using commercially thermoregulated incubators. Refrigerators can be used, but if the door is often open, temperature will vary, impeding reproducibility. Also, vibrations due to the refrigerating compressor can interfere with crystal growth. This drawback can be overcome by dissociating the refrigerator from the compressor. In this chapter, crystallization will be described and correlated with solubility diagrams as described in Chapter 1. Observation is an important step during a crystallization experiment. If you have a large number of samples to examine, then this will be time-consuming, and a zoom lens would be an asset. The use of a binocular generally means the presence of a lamp; use of a cold lamp avoids warming the crystals (which could dissolve them). If crystals are made at 4°C and observation is made at room temperature, observation time should be minimized. Preparation of the solutions of all chemicals used for the crystallization of biological macromolecules should follow some common rules: • when possible, use a hood (such as laminar flux hood) to avoid dust • all chemicals must be of purest chemical grade (ACS grade) • stock solutions are prepared as concentrated as possible with double distilled water. Solubility of most chemicals are given in Merck Index. Filter solutions with 0.22 μm minifilter.
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