Abstract
Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is widely applied for the analysis of complex protein mixtures. Many 2-D PAGE systems and designs are commonly in use. Among these, there is a large group which, for mainly technical reasons, require rod shaped gels for the separation in the first dimension (see e.g. refs. 1-6). Thus, the known advantages of elcctrophorcsis in slab gels. namely sharper and faster resolutions, simultaneous separation of several samples under identical conditions and convenient handling arc not exploited in the first separation step. In this paper, a procedure is described which allows to perform the I -D run in a slab gel. It is demonstrated that the proposed design fits also for those 2-D PAGE systems which normally need rod shaped gels in the first dimension. The widely used slab gel electrophoresis apparatus introduced by Studier [7] can be applied to runs in both dimensions. However, for two reasons a slight modification of the common slab gel cuvette is necessary: (1) Perfectly rectangular stripes containing the separated protein bands have to be cut out of the gel to bc processed for the second run. This operation is not easily carried out, especially when the acrylamide concentration is low (i.e. T = 4%). (2) Considerable lateral diffusion of protein may occur during electrophorcsis in slab gels, especially whcn the protein concentration in a band is high and the adjactent tracks are not used. This may lead to loss of protein when cutting gel stripes of a given size. These problems can bc overcome, when plastic stripes (PVC) are glued (two component adhesive based on epoxy' resin) on that glass plate which normally keeps the slot former, as shown in Fig. l a. The plastic stripes, which are about 0.04 mm thinner than the spacers, serve as excellent guidance when cutting the gel stripes with a razor blade. In addition, lateral diffusion is sufficiently reduced (Fig. 2a, b).
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