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

Protein analysis using low-voltage electrophoresis
Polyacrylamide gel electrophoresis
Disc electrophoresis

Principle of the method. Protein migration and Separation occur in small cylindrical polyacrylamide columns. [The term disc Electrophoresis is derived from the disc-like shape of the gel Column segments containing the fractions, as well as from the English word “discontinuous”, which describes the heterogeneous (“discontinuous”) buffer system typically used in this method.]

Scope of application: fractionation and determination of homogeneity of Proteins (Serum proteins, Enzymes, protein Hormones, etc.), Nucleic Acids, Peptides, etc.

APPARATUS

Several types of apparatus are commercially available. Below we describe the apparatus for analytical Polyacrylamide gel electrophoresis manufactured by Reanal (Hungary).

1. Glass gel tubes and their support stand. Gel columns are prepared in glass tubes with a length of 100 mm and an internal diameter of 6 mm. During gel polymerization, they are inserted into a special stand, which is a double-layer Plexiglas plate with holes drilled in the upper layer for the tubes (Fig. 12, A). The tubes are mounted vertically and secured at the bottom with screw-on Plexiglas rings and rubber gaskets (Fig. 12, B).

2. Electrophoresis apparatus. The apparatus consists of upper and lower buffer reservoirs (Fig. 13) made of Plexiglas. The upper reservoir is mounted on the lower one by Plexiglas posts with screws. A carbon electrode is located in the center of each reservoir inside a cylindrical Plexiglas holder, which, along with the holder, can be removed from the reservoir. Holes are made in the holder wall to ensure continuous contact of the electrodes with the buffer solution regardless of its level, as these holes form a labyrinth system. Twelve glass tubes filled with gel are attached to the bottom of the upper reservoir from below. They are arranged equidistantly from the electrode in a circle with a diameter of 100 mm. Thus, the apparatus allows for the simultaneous analysis of 12 samples. The tubes are attached to the bottom of the buffer reservoir using screw-on rings (Fig. 14).

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Fig. 12. Stand for filling glass tubes with gel, included in the analytical disc electrophoresis kit (see text for description).

Fig. 13. Buffer reservoirs of the analytical disc electrophoresis apparatus.

Fig. 14. Attachment of the glass gel tube to the bottom of the buffer reservoir.

PROCEDURE

1. Preparation of Solutions. a) Stock solutions for gel preparation. Solution А_щ: 36.6 g of Tris (pure) is dissolved in distilled Water, 48 ml of 1 N HCl (analytical grade) and 0.23 ml of N, N, N', N'-tetramethylethylenediamine (pure) are added, and the volume is adjusted to 100 ml with distilled water.

Solution Б_щ: 0.735 g of N, N'-methylenebisacrylamide is dissolved in distilled water, then 28.0 g of acrylamide is added, the volume is adjusted to 100 ml with distilled water, and the solution is filtered.

Solution В_щ: 0.14 g of ammonium persulfate (analytical grade) is dissolved in distilled water and the volume is adjusted to 100 ml.

Solutions А_щ and Б_щ can be stored for several weeks in dark glass bottles in a refrigerator. The shelf life of solution В_щ is no more than one week.

b) Monomer solution for preparing small-pore gel (pH 8.9). Solutions А_щ, Б_щ, В_щ and distilled water are mixed in a ratio of 1 : 2 : 4 : 1. If gel is prepared to fill all 12 tubes, each part corresponds to 3.5 ml.

c) Electrode buffer solution (pH 8.3). 1.2 g of Tris (analytical grade) and 5.76 g of Glycine (analytical grade) are dissolved in distilled water, and the volume is adjusted to 200 ml.

d) Amido Black solution. 10.0 g of Amido Black 10B is dissolved in 7% acetic acid to a final volume of 1000 ml. Before use, the dye solution should be filtered through paper. A 7% acetic acid solution can be used for washing and storing polyacrylamide gel columns.

2. Gel preparation. Glass tubes are secured in the stand. The monomer solution (b) is prepared in a 50 ml vacuum flask, and air is removed from it by creating a vacuum in the flask using a vacuum pump. 2 ml of the monomer solution is pipetted into each glass tube, and to prevent meniscus formation, a 5–8 mm layer of an 8-fold diluted solution А_щ is layered on top. Polymerization occurs in about 30 min. Once it takes place, a sharp boundary forms between the gel and the overlying liquid. After gel formation, the liquid layer is removed with filter paper.

3. Sample application. Test solutions containing approximately 1–2 mg of protein per 1 ml are diluted with an equal volume of 40% sucrose. 50–100 µg of homogeneous or 200–500 µg of heterogeneous protein or protein mixture is applied to each polyacrylamide gel column. After sample application, the tubes are carefully filled with electrode buffer solution. The filled tubes are mounted to the bottom of the upper reservoir, and 1 L of the appropriate buffer solution is poured into each reservoir.

4. Electrophoresis. During the first 30 min of electrophoresis, the current in the circuit should reach 2 mA and should not exceed 5 mA later on. It is recommended to place the operating apparatus in a refrigerator. Separation of serum proteins takes approximately 60–90 min.

5. Removal of the gel from glass tubes. Upon completion of electrophoresis, the tubes are removed from the apparatus and immersed in a water-filled tray. Under water, a narrow spatula or a flexible wire is inserted between the gel and the glass in a spiral motion, peeling the gel from the glass surface. Water penetrates between the gel and the glass, and the gel column is easily removed from the tube.

6. Staining. Gel columns are immersed in the Amido Black solution for 1 h, then rinsed with tap water and transferred to a destaining solution. The destaining solution is changed until the protein-free Regions of the gel become completely transparent.

Notes. 1. Basic PROTEINS AND PEPTIDES are analyzed in an acidic medium (pH 4.3). The following solutions are required for their fractionation.

a) Stock solutions for gel preparation. Solution A_k: mix 48 ml of 1 N KOH (chemically pure) and 17.2 ml of acetic acid (chemically pure), add 4 ml of N, N, N', N'-tetramethylethylenediamine (pure) and bring the volume to 100 ml with distilled water.

Solution B_k: mix 48 ml of 1 N KOH (chemically pure) and 2.87 ml of acetic acid (chemically pure), add 0.46 ml of N, N, N', N'-tetramethylethylenediamine (pure) and bring the volume to 100 ml with distilled water.

Solution V_k: dissolve first 0.4 g of N, N'-methylenebisacrylamide and then 60.0 g of acrylamide in distilled water, bring the volume to 100 ml and filter.

Solution G_k: in distilled water, dissolve first 2.5 g of

N, N'-methylenebisacrylamide, then 10.0 g of acrylamide, bring the volume to 100 ml and filter.

Solution D_k: dissolve 4.0 mg of riboflavin in 100 ml of distilled water and filter before use.

Solution E_k: dissolve 0.28 g of ammonium persulfate (chemically pure) in 100 ml of distilled water.

All solutions except E_k can be stored for several weeks in dark glass bottles in a refrigerator. The shelf life of solution E_k should not exceed one week.

b) Monomer solution for preparing fine-pore gel (pH 4.3). Mix solutions A_k, V_k, E_k and distilled water in a ratio of 1 : 2 : 4 : 1. If preparing gel to fill all 12 tubes, each part corresponds to 3.5 ml.

c) Monomer solution for preparing large-pore gel (pH 6.8). Mix solutions B_k, G_k, D_k and distilled water in a ratio of 1 : 2 : 1 : 4. To avoid spontaneous polymerization after adding solution D_k, the mixture should be protected from light. If it is necessary to fill all 12 tubes of the disc electrophoresis apparatus, one part corresponds to 0.4 ml.

d) Electrode buffer (pH 4.5). Add 16.0 ml of acetic acid (chemically pure) to 62.4 g of ß-Alanine (chemically pure) and bring the volume to 2 L with distilled water.

e) Staining and destaining solutions. The same solutions are used as for the alkaline analysis system (see p. 90).

The gel column is prepared in exactly the same way as for the alkaline analysis system. Then, 0.2 ml of the monomer solution intended for the preparation of the large-pore gel is pipetted onto its top. On top of this, 5–8 mm of an 8-fold diluted solution B_k is carefully layered. After that, the tubes are illuminated for 15–20 min with 400–500 W electric lamps at a distance of 15–20 cm until polymerization occurs, which can be judged by the appearance of gel opalescence. The liquid layer on top is removed with filter paper, and the test solution is applied instead. Unlike the analysis in the alkaline system, the test solution is diluted 8-fold with the diluted solution B_k before application. Otherwise, all steps of the procedure are similar to the alkaline analysis system.

2. One of The most significant advantages of this method is its suitability for studying very small amounts of protein or peptides. This is particularly valuable, for example, for analyzing dilute eluates obtained during Chromatography and Gel filtration. Disc electrophoresis allows these eluates to be analyzed without prior concentration.

3. Stained gel columns can be stored for a long time in test tubes containing destaining solution. Fractionation results are usually recorded photographically, and it is most convenient to photograph the stained columns directly in these tubes.

4. Disc electrophoresis in acrylamide gel containing urea. Solution A: 8 M urea solution (24.0 g of recrystallized urea is dissolved in 50 ml of deionized water, and the pH is adjusted to 3.2 with formic acid). Solution B: 30% cyanogum. Solution C: 0.12 ml of N, N, N', N'-tetramethylethylenediamine (TEMED) in 25 ml of solution A. Solution D: saturated potassium persulfate solution.

Gel preparation. In a vacuum flask, mix 7 ml of solution B, 3.5 ml of solution C, and 17 ml of solution A, and degas the mixture using a vacuum pump. Then, add 0.7 ml of solution D, mix, and fill the glass tubes of the disc electrophoresis apparatus with the resulting solution, as recommended on p. 90. Layer 5–8 mm of distilled water on top of the gel-forming solution.

Fill the buffer reservoirs with a 5.5 M urea solution (330.0 g of urea per 1000 ml of water) adjusted to pH 3.2 with formic acid.

Sample application. Mix the test solution with an equal volume of 40% sucrose and layer it onto the gel. The volume of the applied sample should not exceed 1/₃₀ of the total gel volume.

The electrophoresis and staining Procedures are similar to those already described on p. 90.

References

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Recommended reading

Bailey J. L., Techniques in Protein Chemistry, Elsevier Publishing Company, Amsterdam, London, New York, 1967. (Russian Introduction/27.html">Translation of the 1st ed.: J. Bailey, Methods of Protein Chemistry, Mir Publishers, Moscow, 1965.)

Gordon H., Electrophoresis of Proteins in Polyacrylamide and Starch Gels, North-Holland Publ. Co., Amsterdam, London, 1969.

Leach S. J. ed., Physical Principles and Techniques of Protein Chemistry, Part A, Academic Press, New York and London, 1969.

Smith I., Chromatographic and Electrophoretic Techniques, Heinemann, London, 1969.

Wierne R. J., Agar gel electrophoresis, Elsevier Publ. Co., Amsterdam, 1965.

Williams C. A., Chase M. W., Methods in Immunology and Immunochemistry, Vol. II, Academic Press, New York, London, 1968.

Wunderly C., in: P. Alexander and P. J. Block, A Laboratory Manual of Analytical Methods of Protein Chemistry, Vol. 2, Pergamon Press, Oxford, 1960. (Russian translation: C. Wunderly, in: Analytical Methods of Protein Chemistry, Foreign Literature Publishing House, Moscow, 1963, pp. 151, 167.)