Microscale methods

Two isolation procedures based on methods in category 2 are described in this experiment, a large-scale and a microscale method. Each procedure yields plasmid DNA that is sufficiently pure for size analysis by agarose electrophoresis and for digestion by restriction enzymes as described in Experiment 15. 

The most unpredictable process in X-ray structure determination is the crystallization of the candidate protein into a form suitable for X-ray diffraction. Each protein requires a unique set of conditions to form crystals. Typically 100 mg of highly purified protein is required to determine the conditions that result in usable crystals of 0.1 to 0.3 mm size, although a size of 0.3 to 0.8 mm is preferred. The occurrence of crystals and the rate of crystallization are influenced by many factors such as protein purity, the solvent, concentration of added precipitants, pH, temperature, and the presence of ions and cofactors. The protein solution at a concentration of typically 5 to 20 mg/ml is allowed to slowly reach supersaturation by the removal of or by changing the composition of the solvent by liquid-liquid diffusion or vapor diffusion methods. Microscale methods have been developed to explore several crystallization conditions simultaneously using minimum amounts of the purified protein sample. Recently, use of the zero gravity atmosphere in space has been explored as a means of facilitating crystallization (Eisenberg and Hill, 1990 Branden and Tooze, 1991 Tomasselli et al, 1991). 

Neame, K. D., and Richards, T. G. (1972). Elementary kinetics of membrane transport. BlackweU. Nguyen, R. T., and Harvey, H. R. (1994). A rapid microscale method for the extraction and analysis of protein in marine samples. Mar. Chem. 45, 1-14. 

We have selected material for inclusion in Practical Skills in Chemistry based on our own teaching experience, highlighting those areas where our students have needed further guidance. As a result of our comprehensive cover of practical skills, some techniques such as microscale methods and specialized vacuum techniques have been omitted, but specific references are provided. Instead, we have attempted to provide sufficient detail so that students will have the skills to carry out experiments successfully and not produce poor data as a result of poor technique. 

Until recently, the work in this area has been insuflScient to warrant its review, but some papers on the biochemistry and pathophysiology of connective tissue (F9) are relevant. In consideration of the reports available, it must be borne in mind that comparisons made outside a particular circle of experiments may be invalid on account of the different conditions of the subjects, and conditions and methods for isolation, separation, and measurement of the macromolecules. Many such methods both for tissues and fluids have been reported (see reviews cited in Section 1.1), and it is imperative to ensure that the isolation and separation processes are effective (see B15, K12). Microscale methods have been devised to function on a few micrograms of material for component analysis (e.g., B16 see K17), but more particularly for the eomplete identification of a glycosaminoglycan on a basis of chemical structure (e.g., B13, B14). 

One microscale method developed for differential extraction involves a filter-based system and lysis using acoustic energy. The sample is first infused over a filter (size and material not indicated) in which the sperm cells ( 4-6 [tm diameter) pass through unimpeded and the much larger epithelial cells ( 50 [im diameter) are retained. The DNA is then extracted using ultrasonic disruption of the cells. Although it is too early to gauge the success of this method, filtration has been explored on the macroscale for this application without widespread success. 

The LB [31] is another microscale method that is suited for the efficient treatment of polymer solution dynamics. It has recently been used to investigate the phase separation of binary flttids in the presence of solid particles. The LB method is originated... 

Ribatski et al. [2] presented a comparison between the microscale methods proposed by Kandlikar and Balasubramanian [80] and Zhang et al. [82] and the three-zone method by Thome et al. [85] against a broad database from the literature... 

In general, microscale methods, e.g. formation of mineral precipitates in the pore space of a waste body, will be employed rather than using large-scale enclosure systems such as clay covers or wall constructions (Wiles et al. 1988). An overview on various fields of environmental research and management to which mineralogical methods can be successfully applied has been given by Bambauer (1991). 

Liquid reagents and solutions are added to a reaction by several means, some of which are shown in Figure 7.9. For microscale experiments, the simplest approach is simply to add the liquid to the reaction by means of a Pasteur pipette. This method is shown in Figure 7.9A. In this technique, the system is open to the atmosphere. A second microscale method, shown in Figure 7.9B, is suitable for experiments in... 

In many experiments, it is necessary to remove excess solvent from a solution. An obvious approach is to allow the container to stand unstoppered in the hood for several hours until the solvent has evaporated. This method is generally not practical, however, and a quicker, more efficient means of evaporating solvents must be used. Figures 7.17 and 7.18 show several methods of removing solvents by evaporation. Figure 7.17 depicts microscale methods Figure 7.18 is devoted to large-scale procedures. 

Microscale Methods. A simple means of evaporating a solvent is to place a conical vial in a warm water bath or a warm sand bath. The heat from the water bath or sand bath will warm the solvent to a temperature where it can evaporate within a short time. The heat from the water bath or sand bath can be adjusted to provide the best rate of evaporation, but the liquid should not be allowed to boil vigorously. The evaporation rate can be increased by allowing a stream of dry air or nitrogen to be directed into the vial (Figure 7.17A). The moving gas stream will sweep the vapors from the vial and accelerate the evaporation. As an alternative, a vacuum can be applied above the vial to draw away solvent vapors (Figure 7.17B and 7.17C). 

A convenient water bath suitable for microscale methods can be constructed by placing the aluminum collars, which are generally used with aluminum heating blocks into a 150-mL beaker (Figure 7.17A). In some cases, it may be necessary to round off the sharp edges of the collars with a file in order to allow them to fit properly into the beaker. Held by the aluminum collars, the conical vial will stand securely in the beaker. This assembly can be filled with water and placed on a hot plate for use in the evaporation of small amounts of solvent. 

The ultimate in microscale methods is to use a bulb-to-bulb distillation apparatus. This apparatus is shown in Figure 16.7. The sample to be distilled is placed in the... 

Slurry Method. The slurry method is not recommended as a microscale method for use with Pasteur pipettes. On a very small scale, it is too difficult to pack the column with the slurry without losing the solvent before the packing has been completed. Microscale columns should be packed by the dry pack method, as described in Section 19.6. 

Macroscale Columns. Macroscale columns can also be packed by a dry pack method that is similar to the microscale methods described in Section 19.6. The disadvantages described for the microscale method also apply to the macroscale method. This method is not recommended for use with silica gel or alumina, because the combination leads to imeven packing, air bubbles, and cracking, especially if a solvent that has a highly exothermic heat of solvation is used. 

In the microscale method of determining boiling points, one heats the liquid until a steady stream of bubbles is observed coming out of the bell. The temperature is then lowered and the boiling point is read just as the bubbles stop. Why is this technique preferable to measuring the boiling point when the bubbles first start to appear ... 

A microscale method for detection and quantification of N-acetylneuraminic acid in glycoproteins used acid hydrolysis and HPAEC. Alternatively IV-acetylneuraminic acid released by hydrolysis of serum could be converted into fluorescent compounds (unspecified structures) by reaction with malononitrile, and determined by reversed-phase h.p.l.c. with fluorescence detection. ... 

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