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Search our Tips from A to Z for more information. Still having trouble finding what you want? Contact Hampton Research technical support.
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| C is for Chaotrope |
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| Chaotropic (order-breakers) substances tend to increase the solubility of macromolecules in water. Chaotropic substances decrease the ordered structure of water. Chaotropes lower the cohesion between water molecules. Chaotropes lower the surface tension of water. In general, the surface tension decreases as the chaotropy increases. Chaotropic salts can perturb sample sample and sample solvent interactions by shielding charges and preventing the stabilization of salt bridges. Hydrogen bonding is stronger in nonpolar media, so salts, which increase the chemical polarity of the solvent, can also destabilize hydrogen bonding because there are insufficient water molecules to effectively solvate the ions. This can result in ion-dipole interactions between the salts and hydrogen bonding species which are more favorable than normal hydrogen bonds. Chaotropes break down the hydrogen-bonded network of water, so allowing macromolecules more structural freedom and encouraging protein extension and denaturation. However, lower, non-denaturing concentrations of chaotropes can be used to manipulate sample sample and sample solvent interactions as a means to promote crystallization or improve crystals.
Chaotropes include urea, guanidine, tetramethylammonium chloride. guanidine thiocyanate, thiourea, lithium perchlorate, and sodium thiocyanate.
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| C is for Condensation |
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| Condensation on cover slides or tape may be a problem when incubating plates at cool or warm temperatures.
To minimize condensation try the following:
Place a dummy plate above and below the plates being used for crystallization. Fro example, on a stack of 5 or 10 plates, place a dummy plate on the bottom of the stack as well as the top of the stack. The reservoirs (wells) of the dummy plates should contain water as the reservoir solution. The reservoirs should be sealed either with plain glass cover slides and vacuum grease or clear sealing tape to prevent the reservoir solution from evaporating.
Pipet the reservoirs and drops at the temperature where the plates will be incubated. This means enjoying a cold room during set ups. One can cheat and avoid a cold room by incubating reagents, pipet tips, plates and slides at the cold temperature. When ready to set the experiment set the plates in a ice bath and quickly set the plate while the plate (as well as reagents and protein) are on ice. Conceivably one could also do this for warm temperature incubations where a elevated temperature incubator is available. The idea is to avoid a RAPID change in temperature with the sealed crystallization experiment (plate). By minimizing the temperature difference between the set up and incubation temperatures condensation can be minimized.
Try sitting drop vapor diffusion. Condensation is especially bad for hanging drop vapor diffusion experiments since water condensing on the slide can ÒrunÓ or merges into the crystallization drop. To avoid such a merger try sitting drop vapor diffusion. Condensation on the tape or cover glass above a sitting drop experiment may still make viewing the drop difficult, but at least the drop will not become diluted with condensation unless the condensation is so bad that it rains in your experiment!
Finally, avoid vapor diffusion altogether and go microbatch.
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