Protein Chemistry. Structure, Properties, Research Methods - Shendryk A.N. 2022

Methods for Experimental Study of Protein Structure
Protein Separation Methods
Experimental Methods of Electrophoresis - Paper Electrophoresis

Amino Acids, Peptides, and Proteins can be analyzed by paper Electrophoresis. The best results are obtained using voltages of up to 1 kV.

The electrophoresis Procedure is simple. A strip or sheet of chromatographic paper is evenly moistened with a conductive buffer solution and secured in a specialized chamber between two electrode compartments filled with the conductive solution (see Fig. 4.4).

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Fig. 4.4 Schematic diagram of the apparatus for low-voltage (A) and high-voltage (B) paper electrophoresis.

1 - lid; 2 - paper strip; 3 - Water to maintain constant vapor saturation; 4, 5 - buffer solution Cells; 6 - conductive wick; 7 - cooling plates; 8 - air-filled bag.

The test sample is applied near the edge or in the center of the sheet. Upon applying voltage, the mixture components migrate toward their respective electrodes.

Low-voltage electrophoresis is inefficient for small molecules (such as Amino Acids and NUCLEOTIDES) because their low charge results in low mobility. They separate slowly and undergo significant zone broadening due to diffusion. In high-voltage electrophoresis, the Separation rate increases due to a high potential gradient (200 V/cm). The high voltage causes a large current, which heats the paper, making cooling essential. For cooling, the paper is either immersed in a large volume of an immiscible, non-conductive liquid or pressed against a cooled surface. After electrophoresis is complete, the paper is dried, and the compounds are identified using the same Methods as in low-voltage electrophoresis.

High-voltage paper electrophoresis is particularly valuable for separating amino acids and peptides. Since complete separation of mixtures is not always achieved using electrophoresis alone, it is frequently combined with Chromatography. This technique is known as two-dimensional separation. The sample is first subjected to electrophoresis and then chromatographed at a right angle, or vice versa (see Fig. 4.5).

Fig. 4.5 Combination of electrophoresis and chromatography for the separation of complex mixtures. A - chromatography only; B - chromatography followed by EP

In addition to paper, solid porous Supports such as starch, Cellulose acetate, agarose, and polyacrylamide gel (PAGE) can be used for electrophoresis.