Amino Acids, Peptides and Proteins - Dévényi T., Gergely J. 1976

Protein analysis by low-voltage electrophoresis
Low-voltage paper electrophoresis
Common sources of error in low-voltage paper electrophoresis

1. Poor Separation despite prolonged Electrophoresis. This issue is primarily caused by deficiencies in the buffer solution, possibly due to an excessively high concentration.

2. The boundaries of individual fractions are poorly resolved. This may be due to the low Ionic strength of the buffer solution at a relatively high voltage (see p. 50) or thermal Denaturation of certain Proteins caused by localized heat generation at high voltage.

3. Some protein fractions remain at the point of application. This may be caused by damaging the paper with a pipette during application, or by irreversible binding of some globulins to the paper. The latter can occur if the serum is applied to a dry or insufficiently damp paper strip.

4. Fractions continue to diffuse after the current is turned off. To prevent this, the electrophoregrams should be removed from the electrophoresis chamber immediately after switching off the current.

5. Replicate separations of the same material yield inconsistent results. Poor reproducibility may be due to variations in experimental conditions. To standardize the experimental conditions: a) remember that the same type of filter paper must be used; b) factors affecting electrophoresis must be standardized (such as buffer flow direction, Temperature, etc.) (see p. 52); c) the same dye must be used; d) the method of Quantitative evaluation of electrophoregrams should not be changed. When using the elution method, we recommend staining proteins with acid fuchsin. The unbound fraction of this dye is easily washed out of the paper, while the bound dye is subsequently easily eluted. On the other hand, for photoelectric densitometry of electrophoregrams, staining with Amido Black 10B is highly convenient, as it binds to proteins more strongly than other Dyes and absorbs light approximately 10 times more intensely. Although Amido Black cannot be completely washed out of the paper during destaining, photoelectric measurements after staining with this dye remain sufficiently accurate.