Seeding experiments allow to improve the likelihood of successful protein crystallization and to optimize the growth conditions.
In order to crystallize a biological macromolecule, its concentration is slowly increased until a point of supersaturation is reached. Using the phase diagram,
supersaturation can be displayed in three successive zones, i.e. metastable supersaturation, labile supersaturation and precipitation (Fig. 1). In the metastable zone, no spontaneous nucleation can occur but crystals added to this zone can grow. In the labile zone, spontaneous nucleation takes place and fast growth of the nuclei is observed. In the precipitation zone, the biological macromolecule is many times supersaturated leading to a formation of amorphous precipitate.
Nucleation occurs at a higher level of supersaturation than crystal growth. By placing seeds into a solution supersaturated in the metastable zone, the growth conditions can be optimized and large single crystals can be obtained.
The number of crystals grown can be influenced by the concentration of the seed stock which is added to the protein drop. Seeding with a very concentrated seed stock can result in showering of microcrystals. If the seed stock is too dilute, no nuclei will be transferred to the protein drop. The ideal concentration of the seeding solution can be determined experimentally by performing serial dilutions from a concentrated seed stock.