Section 4.1.4.3. Sample preparation

Preparation of solutions for crystallization experiments should follow some common rules. Stocks should be prepared with chemicals of the purest grade dissolved in double-distilled water and filtered through 0.22 µm membranes. The chemical nature of the buffer is an important parameter, and the pH of buffers, which must be strictly controlled, is often temperature-dependent, especially that of Tris buffers. Commercial PEG contains contaminants, ionic (Jurnak, 1986) or derived from peroxidation, and thus repurification is recommended (Ray & Puvathingal, 1985).

Mother liquors are defined as the solutions that contain all compounds (buffer, crystallizing agent, etc.) at the final concentration for crystallization except the macromolecule. Samples of macromolecules often contain quantities of salt of unknown composition, and it is therefore wise to dialyse new batches against well characterized buffers. Whatever the crystallization method used, it almost always requires a high concentration of macromolecule. This may imply concentration steps using devices operating under nitrogen pressure, by centrifugation, or by lyophilization (notice that lyophylization may denature proteins and that non-volatile salts also lyophilize and will accumulate). Dialysis against high-molecular-weight PEG may also be used. During concentration, pH and ionic strength may vary and, if not kept at the appropriate values, denaturation of samples may occur.