This is an exciting time for all the scientists who are involved in the study of proteins. Although the study of the genome continues intensively, the focus of research switches steadily towards the elucidation of the gene products, the proteins. One of the earliest steps in the study of a single protein, or a complex mixture of proteins is the electrophoresis of the sample, under denaturing or native conditions. The results are crucial as they yield useful information about the molecular weight of the protein, the purity of a sample, the number of proteins that are present following different treatments, the form of a protein under native conditions, etc.
In our lab we isolate proteins from the serum of mice and the supernatants of cell cultures using monoclonal antibodies. The main aim is to obtain a solution pure enough, so we can use it in functional experiments, in order to collect data about the biological significance of the proteins we isolate. I have worked mainly with soluble molecules found in the serum of mice. After the isolation of the proteins with affinity chromatography or immunoprecipitation with magnetic beads, I test the purity of my samples with SDS and Native PAGE using the Vertical Gel Electrophoresis Apparatus, Model V15.17 from Gibco BRL.
The assembly of the apparatus is very easy and when it is completed according to simple guidelines, there are no leakages at all during the approximately 4-hour duration of the electrophoresis. The apparatus comes complete with two sets of glass cassettes, which are thick enough to withstand high pressure during the dismantling of the device. The glasses are accompanied by one set of spacers and a comb. After the assembly of the cassette, the acrylamide solution is poured in the space between the glasses and it takes about 1 hour at room temperature for the gel to form (although an overnight incubation is the best option). After the gel is ready, the glass cassette is placed vertically in the electrophoresis apparatus. The glass of the cassette adjoins to a rubber pad, which is located on the base of the upper tank. Furthermore, two extra rubber pads that are placed on the left and right cleft of the glass cassette protect 100% from any possible leakage. The comb creates wells that can easily accept 50 ìl of sample solution. The upper and the lower tanks of the apparatus need about 1000 ml of running buffer (500 ml per tank), so that the apparatus can function properly. Finally, the glass cassette is protected by a plastic lid that cannot be opened during the electrophoresis. This feature greatly enhances the safety of the whole apparatus.
I operated the device at 120 V and 30 mA. The electrophoresis was performed at room temperate and the heat that was generated was low and very effectively dissipated. At the end of the electrophoresis, I could easily remove the running buffer from the upper and the lower tank using a large 25 ml pipette or by emptying them directly to the sink. Finally, the electrodes could be easily disconnected, washed with distilled H2O and dried, in order to avoid any possible oxidation by the ions that were contained in the buffer.
I have used this apparatus to prepare gels that were stained with Coomassie blue and silver stain. I have also used gels I prepared in the same apparatus for Western blots. Overall, every time I used this device properly, with fresh reagents, the results of the electrophoresis were more than satisfying. I have used the Vertical Electrophoresis Apparatus V15.17 from Gibco BRL, mainly to test the purity of isolated protein samples. Apart from the long duration of the electrophoresis, this apparatus is an excellent companion for everyone who is trying to solve the mysteries of the complex proteome.