Sitting drop method

If the surface tension of the protein solution is rather low one should use the sitting drop method (b) which can be carried out in single drop or multiple drop vessels. Figure kindly supplied by J. Drenth, University of Groningen and reproduced with permission. 

Figure 5.19 Sitting drop method for protein crystallization...

In the sitting drop method a small volume of protein solution is mixed with an equal volume of precipitant solution (which may be a concentrated solution of salts or other precipitants) and placed in a chamber surrounded by a larger volume of the precipitant solution. Water will evaporate from the protein droplet and pass through the vapour phase into the more concentrated precipitant solution. This gradually increases the concentrations of both the protein and the precipitants in the drop, until the precipitant concentration equals that of the surrounding reservoir. Under the right conditions (which are usually achieved by trial and error) this slow increase in concentration (typically over many days) allows growth of well-formed crystals. 

Purified MeHNL was crystallized by the sitting-drop vapor-diffusion method. The 10-20 mm bipyramidal crystals formed were cross-linked with glutaraldehyde and used as biocatalyst for the synthesis of optically active cyanohydrins. The cross-linked crystals were more stable than Celite-immobilized enzymes when incubated in organic solvents, especially in polar solvents. After six consecutive batch reactions in dibutyl ether, the remaining activity of the cross-linked crystals was more than 70 times higher than for the immobilized enzymes. Nevertheless, the specific activity of the cross-linked crystals per milligram protein was reduced compared with the activity of Celite-immobilized enzymes [53],... 

As described earlier, there are a number of different ways of crystallizing proteins. By far, the most common approach is the vapor-diffusion method, as mentioned earlier. Approximately, 70% of the crystal structures reported have been crystallized through variations of the vapor-diffusion method. The technique can be carried out in a number of ways the simplest two being the hanging drop method, and the sitting drop ... 

The main technique employed to set up crystal screens is the vapour diffusion method, either in the hanging drop or sitting drop set up. This method is based on slowly concentrating the droplet solution against a reservoir solution of infinite volmne (ml scale) compared to the volume of the droplet ( xl scale, see Fig. 14.2). Other techniques based on diffusion or counter-diffusion in agarose gels (Biertmnpfel et al., 2002) can also be useful. The... 

To apply the sitting or pendant drop method to measure the surface tension of a liquid the drop must be large enough so that gravitation plays a significant role. Why ... 

The pendant drop method and the sessile drop methods for surface tension appear quite different, but are similar in principle. In each case a drop is formed which either sits on a plane surface (sessile, see Figure 3.10) or hangs as a pendant drop... 

The first crystal structure of CKX, namely ZmCKXl, was published in 2004 [147], Fig. (4A). So far it is the only protein from the number of different enzymes and receptors acting specifically on cytokinins, where the structure has been solved. The crystals of ZmCKXl, expressed in the yeast Pichia pastoris, were crystallized by a sitting drop vapor diffusion method and grown in 100 mM sodium acetate (pH 4.6), 200 mM ammonium sulfate, and 12% (w/v) PEG 5.000 monomethylether. Crystals belonged to the P492i2 space group. 

We therefore describe the basis of macromolecular crystallography and provide a summary of how to understand the results of a crystallographic experiment. We start with a mathematical description of what a crystal means in terms of symmetry this applies to all crystals, whether macromolecular or not. Later, we describe how protein crystals grow by using the hanging drop and sitting drop vapor diffusion methods this explains why protein crystals are so fragile and scatter X-rays very weakly. 

Figure 2.7 The seeding method A small crystal is washed in a series of solutions in which it slowly dissolves, e.g. solutions with a decreasing precipitant concentration. In this way the surface of the crystal is etched and cleaned. It is then transferred to a fresh drop of protein solution with so high a concentration of precipitating agent that the crystal does not dissolve. The drop is then equilibrated with a more concentrated precipitating solution in either the hanging drop or sitting drop mode. Figure kindly supplied by J. Drenth, University of Groningen and reproduced with permission.

Orthorhombic crystals fromRb. sphaeroides RCs are obtained in sitting drops by the vapour diffusion method using polyethylene glycol 4000 (PEG " ) as precipitant and 1,2,3-heptanetriol as additive (Allen... 

Figure 6.10 Sitting Drop-Vapour Diffusion Identical principles to the hanging drop method (see Figure 6.9).

Although microbatch is the simplest method of crystallization, it is a relatively new technique and many experimenters still prefer to use vapour diffusion which has been around and has worked well for over 40 years. Hence, there has also been major development in automating and scaling down the quantities of sample using the popular vapour diffusion method (both sitting and hanging drops). An increasing variety of robots are available commercially. 

Sitting or pendent drop. Both methods involve the determination of the shape of the drop in mechanical equilibrium. The shape is determined by the balance between gravitation and surface tensional forces. If gravitation is negligible the shape is always spherical irrespective of the surface tension. 

---