Basic Instrumentation for Electrophoresis

Instrumentation for performing electrophoresis is both cheap and simple. It consists of a means of generating a DC voltage, a buffer tank which also has some means of holding the separation media between the two electrodes. A schematic diagram of such a horizontal tank is shown in Figure 9. Such an apparatus needs to be levelled in order to prevent the electrolyte from siphoning from one end to the other and so disturbing the electrophoretic resolution. The

Figure 9 Schematic diagram for a paper or membrane elec-trophoresis system.

power supply can be as simple as a 12 V battery, but is usually a mains electricity transformer delivering 100-500 V DC. The required voltage is selected by a digital potentiostat combined with a polarity switch, the run time can also be selected digitally. Usually there are controls to select constant voltage, constant current and constant power modes of operation. If the instrument's designed maxima in any of these modes are exceeded there is a cut-off (safety) trip. Electrical power is usually taken to the electrophoresis tank via a pair of suitably rated copper cables. The tank itself is typically made of a waterproof and electrically insulated plastic material covered with a lid to prevent evaporation and contamination of the system. The size of the tank will depend on the type of separation being undertaken and can range from as small as 100 mm square to as large as 500 mm x 300 mm. The tank is divisible into a number of compartments with plastic spaces. The outer two hold the electrodes. The electrodes are usually made of fine platinum wire connected through the tank wall to the leads from the power supply. Suitable safety devices are included in the tank to prevent operator accidents, for example the power is cut-off if the lid is removed.

With the development of gel electrophoresis system new forms of apparatus were developed. It is necessary to mount and support the gel between the two electrode chambers so that the only electrical connection is via the gel. Electrical contact between the gel and the electrolyte is made either directly or by a wick of some description. Gels can be of many sizes depending on the separation distance required. Tubes of gel with an internal diameter of 1-5 mm and 50-250 mm in length can be prepared, but slab gels, either used vertically or horizontally, are much more common. Vertical slab gels are cast by pouring liquid gel into the space between a pair of glass plates separated by thin spacer strips positioned at the edges and bottom. The plates are clamped to prevent leakage. The thickness of the gel depends on the thickness of the spacers, which can be from 50 |im to 5 mm depending on the application. At the top, a sample well comb is used to create indentations into which samples can be placed. Following the setting and/or polymerization of the gel, the comb is carefully removed and sample(s) (5-10 |L) are loaded into each well. Molecular weight markers, known proteins or DNA fragments, are usually applied to one or both of the two outer lanes. The upper and lower buffer chambers are then filled and the system is connected to the power supply. Appropriate voltages are selected and the power is turned on for the appropriate time. In order to monitor the progress of the separation it is common practice to include a marker dye in the sample. When the dye reaches the end of the gel (normally after some hours) the power is disconnected. The buffer is drained and the gel removed from between the plates prior to staining or blotting (Figure 10).

As well as vertical formats it is also possible to run horizontal slab gels either completely immersed in electrolyte solution (so-called submarine gels) or with suitable wetted connectors.

Although it is relatively easy to construct your own low-voltage electrophoresis system, especially the tanks, systems are available from a large number of suppliers, such as Amersham-Pharmacia, Bio-Rad, Hoeffer, and so on. The cost of commercial systems range from a few hundred pounds to about £5000 depending on size, configuration, maximum voltage and whether or not cooling systems are included. Today, even the preparation of gels can be simplified. Premixed reagents are available to simplify the production of reproducible gels and it is

Figure 10 Schematic diagram of a vertical gel electrophoresis system.

now possible to buy precast gels which only need to be mounted in a suitable holder.

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