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

Protein analysis using low-voltage electrophoresis
Low-voltage paper electrophoresis
Quantitative evaluation of protein separation in low-voltage paper electrophoresis

Quantitative evaluation of stained protein fractions obtained by paper Electrophoresis is based on the fact that the Beer-Lambert law applies to the Dyes used.

There are two Methods for the Quantitative determination of protein-bound dyes: elution of fractions from the electrophoregram followed by colorimetric measurement, and direct spectrophotometry of the uncut strip.

QUANTITATIVE DETERMINATION OF Proteins BY ELUTION: ELUTION OF INDIVIDUAL FRACTIONS

Principle of the method. A section of the paper electrophoregram containing each individual fraction is cut out; the dye adsorbed on this section is eluted and determined colorimetrically.

Procedure. 1. Cutting the electrophoregram into sections occupied by individual fractions. On stained and dried electrophoregrams, the fractions to be eluted are outlined with a pencil. In areas where the boundary between fractions is not clearly visible, i.e., the dye is not completely washed out of the paper, adjacent fractions are divided along the line of minimum staining intensity.

After marking, the sections of the electrophoregram occupied by each individual fraction are cut out. To ensure rapid and complete elution, each section should be further cut into small pieces.

2. Elution. A series of test tubes is prepared According to the number of fractions, and 3 mL of the solution is poured into each. Each section of the electrophoregram corresponding to an individual fraction is placed in a separate tube. Elution is carried out for 2 h at room Temperature, with the tubes being shaken periodically. In the case of alkaline elution, the resulting eluates are acidified with 1 mL of 0.1 N HCl and analyzed colorimetrically to determine the absorbance at the appropriate wavelength.

Table 9 shows the COMPOSITION OF THE eluting solutions, as well as the elution and colorimetry conditions recommended for use in the described methods.

Class="center">Table 9 Elution and colorimetry conditions for different staining methods of electrophoregrams

When the fraction to be determined contains a significant amount of protein (for example, the albumin fraction in serum Protein Electrophoresis), the volume of the eluting solution should be increased to 6 mL in order to obtain absorbance values within the range of maximum instrument accuracy.

3. Quantitative evaluation. Taking dilution into account and Setting the sum of the absorbance values of all fractions as 100%, the percentage of each fraction in the test sample can be calculated.

Notes. 1. The analysis of paper electrophoregrams by the elution method is highly convenient for routine work in clinical laboratories. It is relatively simple, provides rapid results, and offers good reproducibility.

2. The recommended dyes stain serum protein fractions relatively quickly and are protein-specific, meaning they do not stain other serum components. However, different proteins bind dyes with varying affinities. Consequently, artifacts may occur, to eliminate which some authors suggest introducing specific correction factors into the quantitative analysis of electrophoregrams to align the results of zone and free boundary electrophoresis.

Nevertheless, in routine clinical analyses and even in research work, these corrections can be neglected. First, the discrepancy in values may depend not only on the dyes themselves but also on various aspects of the staining, elution, and other techniques. In this case, applying a correction will only partially resolve the error. Second, the analytical technique in the hands of each researcher eventually becomes standardized to such an extent that the results obtained are highly comparable with each other, which fully satisfies the requirements for experiments in routine clinical work. If, however, specific reasons necessitate the Introduction of corrections, their magnitude should be determined under specific experimental conditions, taking into account all the peculiarities of the procedure. Such custom corrections will yield more accurate results than values simply borrowed from the literature.

3. It is convenient to present the results graphically on graph paper. When the width of each fraction, measured on the stained electrophoregram, is plotted on the abscissa, and the percentage of protein of the corresponding fraction is plotted on the ordinate, the distribution of points closely resembles the graph obtained by the Tiselius method. By connecting these points with a curve, we can construct a serum protein profile (proteinogram).

ELUTION METHOD OF EQUAL SEGMENTS OF THE ENTIRE ELECTROPHOREGRAM

Stained and dried electrophoregrams are cut transversely into segments 1–3 mm wide; each segment is eluted individually by one of the methods described in this section and analyzed colorimetrically. On the graph, the absorbance is plotted on the ordinate, and THE POSITION OF the segment on the electrophoregram (in mm) is plotted on the abscissa. Compared to the previous method, this approach yields a more accurate curve. However, it is highly labor-intensive and ultimately does not provide greater accuracy. The percentage of various protein fractions when eluting equal segments of the electrophoregram is determined planimetrically from the resulting curve.

QUANTITATIVE DETERMINATION OF PROTEINS BY THE PHOTOELECTRIC METHOD

Principle of the method: the transparency of the paper electrophoregram is increased using a special clearing agent, and the absorbance of its successive sections is determined in a special instrument.

INSTRUMENTS

1. Vacuum desiccator. A standard laboratory desiccator with a stopcock, connected to a vacuum pump.

2. Densitometer. There are several models of such instruments operating on the principle described below (Fig. 4). The paper electrophoregram, cleared with a special impregnation, is clamped between two Glass plates 1 and placed in front of a slit 2, behind which a light source 3 is positioned. A photocell 4 is located behind the electrophoregram clamped between the glass plates. Based on the current generated in the photocell circuit, the absorbance E can be determined for each section of the electrophoregram:

1) Non-automatic instruments. Densitometers in which the movement of the paper electrophoregram and the recording of absorbance are performed manually.

2) Semi-automatic instruments. Densitometers in which the movement of the electrophoregram and the recording of extinction are performed automatically. The experimenter only needs to perform the quantitative Analysis of the curve.

3) Fully automatic instruments. In these instruments, the movement of the electrophoregram, the recording of extinction, and the calculation of the resulting curve are performed automatically.

PROCEDURE

1. Clearing of the paper electrophoregram. The stained and dried electrophoregrams are immersed in a staining vessel filled with glycerol. Care must be taken to ensure that the glycerol completely covers the paper strips. The vessel with the electrophoregrams immersed in glycerol is placed in a vacuum desiccator, the lid is closed, and a vacuum is applied for 30 min, which facilitates the removal of air bubbles from the paper and better impregnation of the latter with glycerol. If, after 30 min, it appears that the glycerol has not impregnated the electrophoregrams uniformly enough, the entire procedure should be repeated.

Fig. 4. Schematic diagram of an instrument for densitometry of paper electrophoregrams (see text for description).

2. Densitometry of the electrophoregram and recording of the protein profile. The glycerol-impregnated paper electrophoregrams are clamped between two 1 mm thick glass plates supplied with the densitometer. For accurate analysis, it is crucial that no air bubbles remain between the plates and the paper. Bubbles can easily be avoided if, prior to mounting the electrophoregram, the inner surfaces of the glass plates are coated with a thin layer of glycerol. When securing the electrophoregram, the plates are carefully moved relative to each other so that no air bubbles remain between them and the paper. They are then thoroughly wiped with a soft cloth and inserted into the appropriate slot of the densitometer. Responding to changes in extinction, the instrument's recorder plots a curve very similar to the Tiselius curve.

3. Quantitative evaluation of the protein profile. On the protein profile, each fraction is represented by a separate Gaussian curve (Fig. 5). The area under the curve corresponding to each fraction can be measured planimetrically or determined by weighing on a torsion balance a piece of paper cut along its contour.

For weighing, it is always necessary to cut out the entire area outlined by the Gaussian curve of the given fraction. Thus, for example, after weighing the paper section corresponding to albumin, when determining the weight of the section corresponding to α1-globulin, one should add to it the section that is common to the areas of these two fractions. The total weight of all sections is taken as 100%, and based on this, the percentage content of each fraction is calculated.

Fig. 5. Quantitative evaluation of the electrophoretic diagram. The extinction curve obtained using a photoelectric detector (solid line) is resolved into Gaussian curves (dashed lines) corresponding to each fraction. See text for detailed description.

When working with automatic instruments, calculation methods recommended by the manufacturers are usually used.

NOTES

1. For the impregnation of electrophoregrams, the following Reagents can be used instead of glycerol: a mixture of equal parts of liquid paraffin and 1-bromonaphthalene (refractive index 1.5), benzyl alcohol (refractive index 1.54), methyl salicylate (refractive index 1.54).

Nevertheless, glycerol remains the most accessible reagent. It is particularly convenient for impregnating electrophoregrams stained to detect Glycoproteins, but it should not be used when the electrophoregrams are stained with acid fuchsin, as it extracts too much dye from the paper.

The disadvantage of the paraffin-bromonaphthalene mixture is that when it is used, the paper dries rather slowly, and the mixture itself has an irritating effect on the mucous membranes.

2. The Use of appropriate optical filters increases the accuracy of the densitometer. A red filter should be used for densitometry of electrophoregrams stained with blue dyes, and a green filter for electrophoregrams stained with red dyes. (Corresponding recommendations and other operating information are usually contained in the instrument manual.)

3. The percentage values of the fractions determined by densitometry of electrophoregrams show good reproducibility. Naturally, they differ to some extent from the data of free electrophoresis or from the data obtained by the elution method. These discrepancies lead some researchers to introduce special corrections. Our assessment of these corrections is given on p. 61.

4. Clearing of electrophoregrams can be achieved using the "Elphor-Transpavac" device from Bender-Hobein, Germany. The device consists of a cylindrical glass vessel divided into two parts by a constriction. The electrophoregram is placed in the upper part, while the impregnating liquid is in the lower part. The vessel is hermetically sealed with a ground-glass lid, and its upper part is connected to a vacuum pump via a stopcock. Once the required vacuum is reached, the stopcock connecting the vessel to the vacuum pump is closed. The vessel is inverted, and after a few minutes, the stained electrophoregram is impregnated with the liquid. In this case, no air bubbles are formed that would interfere with densitometry.