Equipment and General Methods

Agarose gels (1% w/v) are prepared in the described buffers on glass plates of different size according to need. For example 9x12 cm plates are suited for conventional IE (they permit the analysis of eight samples or seven antisera) and also for RIE (in this case, about 20 samples can be applied). For the CIE and CAIE techniques, 4x9 cm and 7.5 x 10 cm glass plates can be used for the first and the second elec-trophoretic run, respectively. In all cases, one of the surfaces of the glass plates is coated with an agarose solution (1% w/v, in distilled water) that, after being dried, serves as an anchor for the agarose gel. To ensure regular thickness throughout the gel plate, which is essential for the reproducibility of quantitative methods, the melted agarose is poured between two glass plates separated by rigid spacers of around 1.5 mm thickness.

When antibodies, purified proteins or lectins are to be added to the gel, the agarose solution must be previously equilibrated around 55 °C in a thermo-statized water bath to avoid protein denaturation. The growth of microorganisms is avoided by the addition of preservatives to the agarose solutions, for example 0.1% sodium azide or 0.01% sodium merthiolate.

Simple equipment is commercially available, though much can be made in-house. The electrophoresis system only requires two electrophoretic tanks (provided with platinum wire and connected to the electrodes), and an adjustable power supply delivering voltage up to 400 V at 400 mA. The connection between the agarose plates and the buffer in the tanks can be accomplished through filter-paper wicks previously wetted in electrophoretic buffer (the same as that used for preparing the gel). To avoid excessive warming of the gel during electrophoresis, it is convenient to cool the agarose plates using a system connected to tap water. This allows the electrophoresis to be run at room temperature in the laboratory, alternatively, it can be carried out in a cold room at 5°C.

A great number of polyvalent and specific antisera prepared in goat, sheep or rabbits can be obtained from different suppliers or obtained in-house.

Solar Panel Basics

Solar Panel Basics

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