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

Analysis of proteins by low-voltage electrophoresis
Agar gel electrophoresis

Principle of the method: see paper Electrophoresis. Agar gel is used as the supporting medium.

Scope of application: see paper electrophoresis.

EQUIPMENT

1. Power source: see p. 46.

2. Electrophoresis apparatus: see p. 46.

PROCEDURE

1. Preparation of the buffer solution: see p. 47.

2. Purification of agar: see p. 127.

3. Preparation of the agar layer. Glass plates are thoroughly washed, dried, and placed on a strictly horizontal surface (see p. 138).

Two strips of filter paper, 40 mm wide and moistened with buffer solution, are applied to the two edges of the glass plate so that a 10 mm wide edge of the paper is in contact with the glass, while a 30 mm wide section of the paper strip extends beyond the plate.

After this, The surface of the glass plate is coated with a very thin layer of molten agar (1.5% agar in buffer solution). This agar layer should secure the paper strips and prevent the main agar layer, which is applied second, from sticking to the glass.

As soon as the first thin layer of agar solidifies, the main supporting layer of 1.5% molten agar in veronal buffer solution, pH 8.6, is applied to the glass. This layer should be 3–4 mm thick and cover the entire surface of the glass and the filter paper strips. This requires approximately 250–300 ml of molten agar.

Once the agar has solidified, it is trimmed along the contour of the plate and the filter paper strips using a razor blade or a sharp scalpel. For further Processing, the plate is placed on a special support, such as a wooden block or a box approximately 10 cm in width and height. Once the plate is elevated, the paper strips attached to both sides and coated with agar will hang down at a right angle. When securing the plate in the electrophoresis chamber, these strips are immersed in the buffer solution. To ensure uniform electrical conductivity throughout all areas of the agar layer, its integrity must be restored at the folds of the paper strips at the edges of the plate. This is done last, by carefully pipetting molten agar onto any areas where the layer has been damaged.

Before securing the agar plate in the electrophoresis chamber, wells are cut into the agar layer for sample loading. Typically, these wells range in size from 3 to 15 mm and are spaced 12–15 mm apart. They are cut using a blade, a scalpel, or a special glass or metal tool designed for this purpose. The cut pieces of agar are removed by hooking them from underneath or by aspirating them with a syringe equipped with a large cannula.

4. Sample loading. The prepared agar plate is secured in the electrophoresis chamber. The filter paper strips hanging from its ends, which are coated with agar at the top, are immersed in the buffer solution. 0.2 ml of the test serum is diluted with 0.3 ml of distilled Water, warmed to 40°C, and mixed with 0.5 ml of 3% molten agar (prepared in distilled water) cooled to 40°C. Using a pre-warmed pipette, this mixture is poured into the well cut in the agar. After loading the samples, the remaining space in the wells is filled with 1.5% agar prepared in buffer solution. Care must be taken to ensure that the molten agar is completely free of air bubbles.

5. Electrophoresis. At a voltage gradient of 6 V/cm, Blood serum fractionation takes 4–5 h. During electrophoresis, the agar Temperature does not rise above 30°C, so no special cooling devices are required.

6. Protein fixation. As soon as electrophoresis is complete, the filter paper strips hanging from the ends of the plate are trimmed with scissors, and the agar plate is immersed in a 2% acetic acid solution for 1 h. As a result of this procedure, the fixed protein fractions can be clearly observed in the agar.

7. Drying of the agar layer. After fixation, the agar layer is covered with filter paper moistened with distilled water. The paper should be smoothed out over the agar surface to ensure there are no air bubbles between them. The agar layer is then left overnight at 37°C to dry. After drying, the paper is removed, and the agar layer is stained using one of the recommended Methods.

Methods for Staining AGAR ELECTROPHOREGRAMS

Protein Staining with AMIDO BLACK 10B

Staining solution: 1.0 g of Amido Black is dissolved in 900 ml of acetate buffer solution. The acetate buffer solution is prepared by mixing 500 ml of 1 M acetic acid (60 g/l), 500 ml of 0.1 M CH₃COONa ∙ 3Н₂О (13.6 g/l), and 100 ml of glycerol.

Destaining solution: mix 830 ml of distilled water, 20 ml of acetic acid, and 150 ml of glycerol. After staining for 5 h, the agar plates are washed, changing the destaining solution every 20 min, until the protein-free areas of the agar are clear of the dye.

During staining, the agar layer detaches from the glass. After the separated agar plates are stained, they are placed back onto the glass and dried at room temperature.

LIPOPROTEIN STAINING WITH SUDAN BLACK

Staining solution: 1.2 g of Sudan Black is dissolved in a mixture containing 400 ml of absolute alcohol, 380 ml of distilled water, and 20 ml of 25% NaOH solution. The solution is brought to a boil and cooled.

Washing solution: 50% ethanol. After staining for 2 h, the agar plates are washed by immersing them twice for 15 min in the washing solution. If the stained preparation is placed in a 15% aqueous glycerol solution for 5 h, the agar plate detaches from the glass.

Quantitative determination of Proteins in agar gel electrophoresis. The percentage of the resulting fractions can be determined by a photoelectric method. The dried agar plate, detached from the glass, is sufficiently transparent; therefore, without further Treatment, it is placed between the glass plates of a densitometer, and the optical density is measured as described on p. 63.

NOTES

1. Protein migration in the agar gel layer is similar to their movement in free electrophoresis. However, electroosmosis is much more pronounced in agar gel than in paper electrophoresis, which increases the buffer flow toward the cathode. To eliminate potential artifacts, the Sample application wells should be placed exactly in the center of the agar plate.

2. In Grassmann-Hannig type electrophoresis chambers, 16 x 4 cm glass plates can be used. Preparation of the agar layer on these plates is performed in the same manner as described above.

3. Instead of introducing the protein solution (e.g., blood serum) into a pre-cut well, it can be applied to a 10 x 3 mm strip of filter paper, which is then placed on the agar surface at the appropriate Location, allowing the protein solution to diffuse into the gel. The advantage of this method is that it eliminates the need to cut wells, which would otherwise disrupt the integrity of the electrophoregram. On the other hand, this application method does not allow for an accurate Determination of the amount of protein that has diffused into the agar.

4. During electrophoresis, the migration of serum Proteins can be directly observed if they are stained with Congo red or bromophenol blue. These Dyes migrate along with the albumin fraction.

5. In addition to the described fixative, a fixing solution of the following composition can also be used for lipoprotein staining [29]: a mixture of 500 ml of ethanol, 20 ml of 10% CaCl₂ solution, and 20 ml of acetic acid, with the volume adjusted to 1000 ml with distilled water. The agar plates are placed in this fixative for 6 h.