ANALYTICAL BIOCHEMISTRY 70, 612--613 (JS,/o)

SHORT COMMUNICATIONS An Apparatus for Microelectrophoresis in Polyacrylamide Slab-Gels Maurer and Dati (1) have described a simple method by which several samples containing microgram quantities of protein may be subjected to electrophoresis in parallel within a polyacrylamide slab-gel. The apparatus described here, which is essentially a miniature version of that of Reid and Bielesky (2) appears to be more convenient to use than Maurer's. A particular advantage of the new apparatus is that the upper surface of the gel slab is easily accessible after the gel has been connected to the electrode chambers. Samples can be loaded at this stage, and there is so little subsequent disturbance that a plug gel is not necessary to keep them in place.

MATERIALS AND METHODS Apparatus. The apparatus consists of a glass chamber (Fig. 1) which contains the gel and a holder (Fig. 2a-c) to which the chamber is attached during the run. The holder is shaped so that current can be passed between an upper and a lower electrode bath via the gel. It is essential to

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FIG. 1. (a), Exploded diagram of the gel chamber, which is made of glass. Two strips of glass, 1.2 mm thick, are cemented to the plate on the left as spacers. The bottom spacer is removable. The plate on the right is not large enough to cover the upper region of the spacers when the chamber is assembled. Two squares of silicone rubber sheet of approximately the same thickness as the glass are attached to the uncovered regions by means of silicone grease. (b) Plan diagram of the assembled chamber. Dimensions are in millimeters. 612 Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

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F]G. 2. (a-c) Plan and elevations of the gel holder, which is made from l-in.-thick clear Perspex (Lucite). The two holes shown are tapped to received miniature sockets. (d) Comb, also made of Perspex. Dimensions are in millimeters.

make the gel chamber entirely of glass since polyacrylamide does not adhere well to Perspex. The glass plates for the front and back of the chamber are cut from 38 x 76-ram microscope slides. Note that they are dissimilar in size (Fig. lb). Two glass strips are cemented to one of the glass plates as spacers. The glass used was 1.2 mm thick. The base of the chamber is formed by a third, removable glass strip of the same thickness. When the chamber is assembled the glass plates are smeared with a very small quantity of silicone grease and clamped together by means of paper clips at the sides. The comb (Fig. 2d) is made so that the teeth fit snugly into the chamber. Its purpose is to form indentations in the gel into which the samples are loaded. Holder and comb are made from clear Perspex according to the plan shown in Fig. 2a-c. Electrodes of platinum wire, 0.5 mm in diameter, are soldered into miniature sockets, and the wires are bent to dip into the electrode baths as shown in Fig. 3. M e t h o d . For a discontinuous sodium dodecyl sulphate (SDS)-gel system using, for instance, the solutions described by Laemmli and Favre (3) the procedure is as follows. Separation gel solution, 0.8 ml, is pipetted into the assembled chamber and carefully overlayered with 0.01% S DS solution by using a small hand-held pipet. When the gel has polymerised its surface is rinsed with buffer and the gel chamber is filled to the brim with stacking-gel solution. The Perspex comb is then inserted. When

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FIG. 3. Holder with gel chamber attached by means of paper clips. The design shown here is slightly different from that shown in Fig. 2 in that the lower electrode bath is smaller. t h e s t a c k i n g gel h a s p o l y m e r i s e d t h e gel c h a m b e r is c l a m p e d a g a i n s t t h e h o l d e r as s h o w n in F i g . 3. S m a l l s q u a r e s c u t f r o m a s h e e t o f s i l i c o n e r u b b e r ( s h o w n s t i p p l e d in F i g s . I a n d 3) a r e i n s e r t e d so t h a t t h e s o l u t i o n c a n n o t flow o u t f r o m the u p p e r e l e c t r o d e b a t h . T h e l o w e r b a t h is filled b e f o r e

FIG. 4. Microslab gel. Scale bar = 10 mm. The gel contains proteins which have been separated in a 15% polyacrylamide discontinuous SDS gel and stained with Coomassie blue (3). Five-tenths microgram of each of the standard proteins, bovine serum albumin (BSA), carbonic anhydrase (CA) and lysozyme (L), has been loaded. The other two samples each contained protein from 15 contractile organelles from the protozoan Zoothamnium.

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the strip is removed (by means of a needle) from the bottom of the gel chamber. Note that the strip is made a little longer than the width of the gel chamber to facilitate this (Fig. 1). The optimum sample volume is 5/zl, which can easily be loaded with a fine hand-held pipet. When handling these small volumes it is useful to add 0.02% fluoroscein which can be made to fluoresce brilliantly with the light from a quartz-halogen bulb. The fluoroscein serves as tracking dye during the run. At 8 mA the run time is 20 min, staining in Coomassie blue takes 30 min at 50-70°C, and destaining, using heated solution, is complete in a further 30 rain. The resulting gel, which measures 30 × 50 ram, can be scanned directly with a microdensitometer.

RESULTS AND DISCUSSION The apparatus was devised in order to analyse the protein composition of small numbers of contractile organelles individually dissected from a protozoan (4). Figure 4 shows a 15% acrylamide-SDS gel in which the proteins from the organelles have been separated in parallel with a sample of standard proteins. The finest bands visible represent 20-100 ng of protein. As well as high sensitivity, the slab-gel system has the advantage that concentrated gels can be removed easily from the holder. As pointed out by Maurer, a further increase in sensitivity could be achieved by using a smaller slab, but micromanipulation would then be needed.

REFERENCES 1. 2. 3. 4.

Maurer, H. R., and Dati. F. A. (1972)Anal. Biochem. 46, 19-32. Reid, M. S,. and Bieleski, R. L. (1%8) Anal. Biochem. 22, 374-381. Laemmli, U. K., and Favre, M. (1973) J. Mol. Biol. 80, 575-599. Amos, W. B., Routledge, L. M., and Yew, F. F. (1975) J. Cell Sci. 19, 203-213.

W. B. AMOS University of Cambridge Department ~]" Zoology Downing Street Cambridge CB2 3EJ England Received Januao' 7, 1975: accepted September I1, 1975