A high resolution gel densitometer based on the Zeiss PMQ II microcuvette chamber

Abstract

The optical evaluation of the peptide band pattern in polyacrylamide gels is essentially possible by direct densitometry or by densitometry of photographs of the gels. The implications of either method and of gel densitometry in general are beyond the scope of this paper (1,2). High performance systems for direct gel densitometry should be able to resolve bands as thin as 0.05 mm and only 0.05 mm apart (1); determination of the optical density should be possible with sufficient precision at the absorption maximum of the dye-protein complex. Methods that can cope with these demands are normally based on a microscope or a photometer [(4); for a survey see (1,2)] and require elaborate optical equipment, monochromator, lightdetector, a recording device, and a system for precise linear transport of the gel. Consequently commercial gel densitometers with high resolving power are expensive and therefore not available in every laboratory. The type of commercial densitometers for clinical use, recommended by the manufacturer for scanning of polyacrylamide gels, inevitably lead to poor resolution as compared to direct yisual inspection. This is mainly due to the rather simple optical equipment of these instruments. In addition only very few commercially available densitometers, whether of high performance or not, are apt to handle very long gels (2030 cm). Yet only gels of this length allow to make full use of the separation potential of SDS-disc electrophoresis (3). We developed a simple method to scan polyacrylamide gels with a favorable degree of resolution by combining the high performance optical system of the microcuvette chamber of the Zeiss PMQ II spectral photometer, which is still in use in many laboratories, with a glass tube as cuvette and an effective transportation device for long cylindrical gels. The optical equipment of the microcuvette chamber of the Zeiss PMQ II is designed to allow measurements in cuvettes with extremely long and narrow light passes. This is achieved by combined focusing and collimation of the light beam. A simple and easily built transportation device consists of a long glass tube filled with liquid and a low-pulse peristaltic pump as normally used for column chromatography. The gel moves in the glass tube like a piston in its cylinder. Figure 1 shows a schematic drawing of the whole arrangement. Holes of 9.5 mm diameter are drilled through the side walls of the microcuvette chamber (A) at the positions marked with a cross in Fig. lb, after the microcuvette holder has been removed from the sample changer rod. These holes do not forestall further normal use of the microcuvette chamber; if necessary, they may be closed with rubber stoppers. A glass tube of 60 cm length and 6.0 mm inner and