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Crystallization Techniques
Batch Crystallization This is the oldest and simplest method of crystallization. The principle is to simultaneously add a precipitant to the protein solution and immediately bring the solution to a high supersaturation state. Fortunately, crystals can be gradually grown in a supersaturated solution without further processing. An automated system for differential batch crystallization has been designed by Chayen et al. (1991, 1992) in which they grow crystals in 1-2 [mu]l droplets containing protein and precipitant. The droplets are suspended in an oil such as paraffin. The oil acts as a seal to prevent evaporation. It does not interfere with common precipitants, but interferes with organic solvents that dissolve the oil.
Liquid-Liquid Diffusion In this method, the protein solution and the solution containing the precipitant are layered in each other in a capillary having a small pore, and a capillary for measuring the melting point is generally used. The lower layer is a dense solution such as concentrated ammonium sulfate or a PEG solution. If an organic solvent such as MPD is used as a precipitant, it will be in the upper layer. With a 1:1 mixture, the concentration of the precipitant should be twice the final concentration. The two solutions (about 5 μl each) were introduced into the capillary through a syringe needle and introduced into the lower layer first. The bubbles are removed by a simple rocking centrifuge. Then add the upper layer, and then form a distinct interface between the two layers, they will gradually spread to each other. Garc'?a-Ruiz and Moreno (1994) have developed liquid-liquid diffusion techniques to acupuncture methods. The protein solution is drawn into the narrow tube by capillary force, and one end of the tube is closed. The open end is then inserted into a gel placed in a small container, the gel making the tube vertical and the protein solution in contact with the gel. The solution containing the precipitant was poured onto the gel and the entire unit was stored in a closed box to prevent evaporation. The diffusion time of the precipitant through the gel and capillary can be controlled by the depth of the capillary inserted into the gel, so that a supersaturated region can be formed in the protein solution, with the bottom of the capillary being high and the top being low. This can also be used as an additional information to screen for optimal crystallization conditions.
Vapor Diffusion
The Hanging Drop Method In this method, droplets are prepared by mixing 3-10 μl of protein solution and an equal amount of precipitant solution on a siliconized microscope cover slip. The coverslip is placed over the recess of a plate with a portion of the recess filled with about 1 ml of the desired precipitant solution. The groove around the chamber is sealed with oil or grease before the coverslip is placed.
The Sitting Drop Method in the hanging drop method, if the surface tension of the protein solution is small, it will spread on the surface of the coverslip. At this point, the drop method is more advantageous.
Dialysis In addition to the above methods for crystallizing proteins, there are many dialysis techniques. The advantage of dialysis is that the precipitation solution is easily changed, and for a suitable amount of protein solution (more than 0.1 ml), it can be done with a dialysis tube. The dialysis membrane is attached to the tube via a rubber band, but is rinsed with plenty of water or preferably in water for about 10 minutes before use. For microliters of protein solution, a thick-walled microcapillary (Zeppezauer method) coated with a dialysis membrane or a plexiglass button can be used. The disadvantage of the buttons is that the protein crystals in the buttons cannot be observed by a polarizing microscope.
In another microdialysis method, 5 μl of the protein solution was injected into a capillary tube covered with a dialysis membrane, and the membrane was tightly packed with a plastic tube. The protein solution was briefly centrifuged, and then the capillary was closed with mold clay, and then the capillary was placed in an Eppendorf tube containing dialysate.
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