Vertical electrophoresis

Wait for 5-lOmin and let the gel solidify. Then, remove the combs slowly and assemble plates containing the gel in the designed place in the vertical electrophoresis apparatus. 

Equipment PCR machine, scintillation counter, tabletop centrifuge, temperature-controlled water baths, equipment for horizontal and vertical electrophoresis, UV-illuminator, phosphor imager, automatic DNA sequencer, vacuum dot-blot manifold (Schleicher and Schuell). PCR 0.5 ml hot-start mbes, aerosol resistant pipette rips, autoclaved Eppendorf tubes (all from Fischer Scientific, Brightwaters, NY) and glassware, diethyl pyrocarbonate (DEPC, Sigma)-treated solutions. 

Fill the gel particles together with the surrounding buffer into the tube. The tube is inserted into the vertical electrophoresis apparatus (dialysis membrane down to anode), electrode buffer is poured, and electroelution is started with 10 mA per tube for 3-8 h, depending on tbe molar mass of the protein. 

A vertical electrophoresis apparatus for a slab gel. Courtesy of Hoefer Pharmacia Biotech, Inc., San Francisco. 

Vertical electrophoresis systems (Amersham Pharmacia Biotech, Piscataway, NJ, USA) Trans-Blot semi-dry transfer cell (Bio-Rad, Hercules, CA, USA) hybridization incubator (Fisher, Hanover Park, IL, USA) personal densitometer SI (Molecular Dynamics, Sunnyvale, CA, USA). Additional materials and equipment needed are described throughout the text. All chemicals are of reagent grade. 

Prepare a polyacrylamide separating gel of appropriate concentration, according to Table 1, in a vertical electrophoresis apparatus. After the separating gel has set, cast a 4.5% polyacrylamide stacking gel on top (see Table 1), with a comb to form sample wells. 

Fig. 7.10. Flat-gel vertical electrophoresis apparatus of the Akroyd type, showing three gels being run in parallel (De Wachter and Fiers 1971).

Fig. 7.11. Flat-gel vertical electrophoresis apparatus a more complex design, adaptable for the utilisation of several flat gels simultaneously. A is the anode, B an inlet for circulation of buffer in the upper reservoir, C, D the sample slot former, E the electrode, F the gel, H a sUicone rubber gasket, J the lower reservoir and K the outlet for buffer circulation from the lower reservoir (by courtesy of Universal...

Fig. 7.12. Flat-gel vertical electrophoresis apparatus after Raymond (1962), and manufactured by E-C Apparatus Co. The gel is supported on a sponge strip, F, and is confined between two cooling plates A and B. The samples are applied in slots, E, C and D are electrodes. G the reservoir buffer levels, and H a constant-level tube.

Equipment tips and filtertips, waterbath, heating block, microcentrifuge and tubes, horizontal (Bio-Rad) and vertical electrophoresis system (SE250 Mighty Small II, Amersham Pharmacia Biotech), power supply, UV transilluminator and... 

Western blotting (Towbin, H., Staehelin, T., and Gordon, J., Proc. Nat. Acad. ScL, U.S.A., 76, 4350, 1970) is designed to detect proteins. The gel is a thin layer of polyacrylamide and vertical electrophoresis is used. The membranes are nitrocellulose or polyvinylidene difluoride (PVDF). Usually an antigen-antibody reaction (immuno-blotting) is used for detection. 

Figure 27-25. A vertical electrophoresis apparatus, EDVOTEK Model MV 10.

Fig. 6.2. Hanging strip type apparatus (vertical electrophoresis cell) (according to Ref. 8).

These volumes will produce sufficient solution to pour four 8 cmxlO cm separating gels of 1.5 mm thickness in GE Healthcare Life Sciences SE250 and SE260 Mini-Vertical Electrophoresis units... 

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