Process electrophoresis

Cover Image Gel used in electrophoresis process to separate proteins (biochemistry). Photograph by Peter Poulides. Reproduced courtesy of Tony Stone Images. 

B 8. In this experiment, you used bromophenol blue dye as a marker to tell you when to stop the electrophoresis process. What assumptions must you make about the relative movement of the dye versus sample proteins during electrophoresis ... 

Low concentrations of proteins found in ES products are further complicated by high concentrations of salts. Salt contamination directly interferes with the electrophoresis process. The movement of salt through the first dimension to the extremities of the pH gradient will prolong the time needed to separate... 

The use of SDS is not always without drawbacks. One of the most important is encountered when the sample is rich in DNA. A terrible viscosity results, which can hamper the electrophoresis process. Moreover, some protein classes (e.g. glycoproteins) bind SDS poorly and are thus poorly separated in the subsequent electrophoresis. In such cases, it is advisable to use cationic detergents. They are usually less potent than SDS, so that a urea-detergent mixture must be used for optimal solubilization (MacFarlane 1989). Moreover, electrophoresis in the presence of cationic detergents must be carried out at a very acidic pH, which is not technically simple but still feasible (MacFarlane 1989). This technique has however gained recent popularity as a double zone electrophoresis method able to separate even membrane proteins (Hartinger et al. 1996), and showing more separation power than SDS electrophoresis alone. 

During the electrophoresis process, the molecules migrate in the gel under the influence of an applied electric field. The distance the compounds can travel in the gel depends on several factors, which are as follows ... 

This is the single most critical procedure in the whole electrophoresis process. The sample may be applied as a spot (about 0.5 cm in diameter) or as a narrow uniform streak. Special devices are available commercially for this purpose. There is no hard and fast rule for the time of application of the sample to the electrophoretogram. This can be applied before the paper has been equilibrated with the buffer or after it. 

CFE separator (Figure 7.3.4) is strictly nonuniform. The electrical field varies inversely with the radius (see Problem 3.1.5) the electrophoretic radial migration velocity therefore decreases as the radial coordinate of the charged solute molecules increases. Due to the very small thickness of the annular region in the rotationally stabilized CFE separator, the electrical field is, however, almost uniform. Rolchigo and Graves (1988) have modeled continuous electrophoresis processes in general when subjected to a nonuniform electrical field. 

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