Hanging-drop technique, crystallization

It proved possible to crystallize the Fepr protein over a wide range of pH, 5.9-8.0, and PEG 8000 concentrations, using both sitting and hanging-drop techniques. The crystals used for the X-ray analysis were produced using the following procedure (29) ... 

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 ... 

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. 

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... 

The technique of choice for screening crystallization conditions is the vapour diffusion hanging drop described in Chapter 3. in this method drops containing different concentrations of protein-DNA mixtures, buffer, additive, and precipitant are suspended from a sihconized coverslips placed over sealed reservoirs containing different precipitant... 

One widely used technique is vapor diffusion, in which the proteinlprecipitant solution is allowed to equilibrate in a closed container with a larger aqueous reservoir whose precipitant concentration is optimal for producing crystals. An example of this technique is the hanging-drop method (Fig. 3.2). 

The Raman spectrum shown in Figure 18-10 illustrates how a fiber-optic probe can be used to monitor chemical processes. In this ca.se a fiber-optic probe w-as used to monitor the hanging drop crystalli/ation of aprotinin (a serine protease inhibitor) and (Nll4)-S04 in aqueous solution. Kaman bands were attributed lo both the protein and the salt. By using chemomet-ric techniques, changes in the spectrum during crystallization were correlated with depletion of both the protein and the salt. The authors were able to determine accurately supersaturation of aprotinin using this technique. 

Crystallization procedures are carried out using the hanging-drop vapor diffusion technique. The droplet (typically 4 p.1) is prepared by mixing equal volumes (2 p,l) of the protein and reservoir solutions. ... 

Hanging-drop Crystallization Another common protein crystallization technique is the hanging-drop crystallization method, where a drop of protein in precipitation solution is suspended above a reservoir until crystallization is realized. This protocol was mimicked in a recent study in which the protein/precipitant solution was suspended from a Ge ATR crystal [99]. The resulting images obtained allow discrimination between protein adsorbate and salt crystal formation (Figure 9.21). In situ micro-ATR FT-IR imaging of the protein crystallization... 

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