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

Protein analysis by low-voltage electrophoresis
Low-voltage paper electrophoresis
Staining procedure

Various staining techniques for heat-fixed electrophoretograms allow not only the detection but also the quantitative measurement of different protein fractions. In addition to Protein Staining Methods, There are also techniques for staining the lipid and carbohydrate components of Lipoproteins and Glycoproteins.

PROTEIN STAINING

Protein Staining with AMIDO BLACK

1) Purification of Amido Black [14]. Dissolve 10.0 g of pure Amido Black in 900 ml of distilled Water, and then precipitate it by adding 100 ml of concentrated Hydrochloric acid. Decant the supernatant and wash the precipitate in 1000 ml of 10% HCl, then place it on a filter and wash with cold distilled water until the blue color disappears from the filtrate. Next, transfer the dye precipitate to a beaker, suspend it in distilled water, and adjust the pH of the suspension to 6.5 using NaOH. After that, heat to boiling, cool, check the pH, and dry on a water bath. Use the dye purified in this manner to prepare the staining solution.

2) Staining according to Grassmann and Hannig [11] (see p. 48).

3) Staining by the method of Putra [23]. Staining solution: dissolve 1.0 g of Amido Black in a mixture of 90 ml of methanol and 10 ml of glacial acetic acid.

Washing solution: 5% acetic acid.

Procedure. Roll up the fixed electrophoretograms, immerse them for 2–3 min in methanol heated to boiling on a water bath, and then stain for 4 min with the Amido Black solution at 80°C. After staining, flatten the strips on Glass, leave for 5 min, and then wash in the washing solution heated to 80°C until the excess dye is removed.

PROTEIN STAINING WITH ACID FUCHSIN

Staining solution: dissolve 2.0 g of acid fuchsin in a mixture of 500 ml of methanol, 400 ml of distilled water, and 100 ml of glacial acetic acid.

Differentiating solution: mix 500 ml of methanol, 400 ml of distilled water, and 100 ml of glacial acetic acid.

Washing solution: 10% acetic acid.

Procedure. Immerse the heat-fixed electrophoretograms in the staining solution for 15 min, then transfer them to the differentiating solution for 15 min. After that, wash the electrophoretograms, changing the washing solution every 20 min, until the protein-free areas of the paper are completely free of dye.

The ability to completely remove unbound fuchsin from the paper is an advantage of this method, making it particularly convenient for the Quantitative determination of fractions by elution (see p. 60). The same portion of staining or differentiating solution can be used 4–5 times. For the initial washes of a new series of electrophoretograms, the final portions of the washing solution from the previous staining procedure can be used.

PROTEIN STAINING WITH BROMOPHENOL BLUE [3]

Staining solution: 1% solution of bromophenol blue in ethanol saturated with HgCl₂.

I differentiating solution: methanol containing 1% HgCl₂.

II differentiating solution: ethanol containing 1% HgCl₂.

Washing solution: methanol.

Procedure. Immerse the fixed electrophoretograms in the staining solution for 5 min, transfer them to differentiating solution I, and then to differentiating solution II for 15 min. After that, wash the paper with pure methanol. If necessary, the electrophoretograms can be dried very quickly by immersing them in ether and exposing them to air. Instead of methanol, 0.5% acetic acid can be used as the washing solution.

PROTEIN STAINING WITH PONCEAU RED [24]

Staining solution: saturated solution of Ponceau Red 2R in a 50% methanol – glacial acetic acid mixture (9:1).

Washing solution: 50% methanol – glacial acetic acid mixture (9:1).

Procedure. Staining is carried out for 10 min, then the electrophoretograms are washed until the unbound dye is completely removed.

PROTEIN STAINING WITH AZOCARMINE [17]

Staining solution: 0.25% solution of azocarmine in a methanol–acetic acid mixture (9:1).

Washing solution: methanol–acetic acid mixture (9:1).

Procedure. Staining is carried out for 10 min, then the electrophoretograms are washed until the unbound dye is completely removed, changing the washing solution every 15 min.

LIPOPROTEIN STAINING

LIPOPROTEIN STAINING WITH SUDAN BLACK (Modification of the METHOD USED IN OUR LABORATORY)

Stock dye solution: 500 mg of Sudan Black 10B is dissolved in 500 ml of methanol.

Staining solution: to 100 ml of the stock dye solution, add 180 ml of methanol and 120 ml of distilled water.

Procedure. Heat-fixed electrophoretograms are immersed in the staining solution for 60 min and then washed in running tap water until the unbound dye is completely removed.

The stock and staining solutions should be stored in the dark in tightly closed bottles and must be filtered through paper before use. It is best to use the stock solution not immediately after preparation, but after letting it stand for 6–8 days. The same portion of the staining solution can be used 4–5 times. After that, the dye precipitates, the dark blue tint of the solution turns black, and upon filtration, the dye granules remain on the filter.

Sudan Black 10B stains lipoproteins blue. Usually, the Background, i.e., the areas of the electrophoretogram containing no lipoproteins, cannot be washed to an absolutely white color because the dye cannot be completely removed from the paper. This should be kept in mind when determining lipoprotein content by the elution method (see p. 60).

LIPOPROTEIN STAINING WITH SUDAN BLACK ACCORDING TO SWAHN [28]

Stock dye solution: 0.1 g of pure Sudan Black is dissolved in 100 ml of 60% ethanol while heating on a boiling water bath. After cooling, the dye solution is filtered twice through a fine-pore paper filter.

Procedure. Electrophoretograms are immersed in the staining solution for 3 h, then washed for 45 min in three changes of 50% ethanol and air-dried.

LIPOPROTEIN STAINING WITH FAT RED [12]

Staining solution: 0.4 g of pure Fat Red is dissolved in 1000 ml of 60% ethanol, heating to boiling in a flask with a reflux condenser. The mixture is then left overnight at 30°C on a magnetic stirrer. Finally, the solution is filtered at 39°C.

Procedure. Heat-fixed electrophoretograms are immersed for 18 h in the staining solution heated to 30°C, then washed with running tap water for 2 min to remove ethanol, and dried at room Temperature. Staining at temperatures below 30°C leads to precipitation of the dye. Lipoproteins are stained red against a pink background.

DETECTION OF LIPOPROTEINS BY Electrophoresis OF PRE-STAINED SERUM [5]

To 1 ml of serum in a Wassermann tube, slowly add 0.1 ml of a saturated solution of acetylated Sudan Black B in 95% ethanol, mix thoroughly, and leave for 30 min. Then, remove the excess ethanol by blowing air over the serum. During staining, precipitation of the dye may occur, which is easily removed by centrifugation.

8 μl of the stained serum is applied to a strip of filter paper and subjected to electrophoresis in Michaelis buffer, pH 8.6, μ = 0.05. Well-separated fractions of α- and β-lipoproteins form blue-colored zones against the white background of the electrophoretogram.

Notes. On Macherey-Nagel 214 paper at a voltage of 300 V in a zonal semi-microelectrophoresis apparatus, lipoproteins can be completely separated in 1.5 h.

Acetylation of Sudan Black 10B [19]. To 60 ml of pyridine, add 40 ml of acetic anhydride. Add 2.0 g of Sudan Black 10B to this mixture and leave overnight. Then, bring the volume of the mixture to 3 L with distilled water, collect the precipitated dye, dry it, and dissolve it in acetone.

GLYCOPROTEIN STAINING

Serum glycoproteins are detected with toluidine blue, which yields metachromatic staining with them, or by a color reaction with periodic acid and Schiff's reagent (PAS reaction).

GLYCOPROTEIN STAINING WITH TOLUIDINE BLUE [2]

Reagents. 1) 1.2 g of periodic acid is dissolved in 30 ml of distilled water, and 15 ml of 0.5 M sodium acetate solution and 100 ml of 96% ethanol are added. This reagent should be prepared immediately before use.

2) Differentiating solution: to 100 ml of methanol, add 20 ml of glacial acetic acid and 80 ml of distilled water.

3) Bromine water.

4) 1% aqueous solution of toluidine blue.

5) 4% ammonium molybdate solution.

6) Acetone.

Procedure. Heat-fixed electrophoregrams are immersed in freshly prepared reagent (1) for 15 min, then transferred to bromine water (3) for 15 min. After this, the electrophoregrams are rinsed with tap water and immersed in toluidine blue staining solution (4) for 30 min. Then, the staining solution is drained, and the electrophoregrams are washed with running tap water for 30–40 min until the unbound dye is completely removed. Next, they are fixed in solution (5) for 3 min and immersed in differentiating solution (2) for 15 min. As a result, blue glycoprotein fractions are clearly visible against a reddish-violet background. Staining is completed by a two-minute immersion in acetone and air-drying.

GLYCOPROTEIN STAINING WITH PERIODIC ACID – SCHIFF REAGENT [16]

Reagents. 1) Dissolve 1.2 g of periodic acid in 30 ml of distilled water, add 15 ml of 0.5 M sodium acetate solution and 100 ml of ethanol. Prepare the reagent immediately before use.

2) Dissolve 5.0 g of potassium iodide and 5.0 g of sodium thiosulfate in 100 ml of distilled water, and add 150 ml of 96% ethanol and 2.5 ml of 2 N hydrochloric acid. This solution is also prepared immediately before use.

3) Dissolve 1.5 g of basic fuchsin in 200 ml of distilled water, add 1.5 g of potassium metabisulfite and 3 ml of concentrated HCl. Leave the solution in the refrigerator for 12 h, then mix it with 3–4 g of charcoal powder and filter through a dense paper filter. Repeat filtration until the filtrate is almost completely decolorized (a light pink tint is acceptable). The prepared solution can be stored for 2–3 days.

4) Dissolve 0.4 g of potassium metabisulfite in 10 ml of distilled water and add 1 ml of concentrated HCl.

5) Add 3 parts of concentrated HCl to 100 parts of 40% formaldehyde solution.

Procedure. Fixed electrophoregrams are immersed in reagent (1) for 10 min and then washed in 70% ethanol. Then they are immersed in reagent (2) for 8 min and washed again in 70% ethanol. After that, they are stained with reagent (3) for 30 min and washed three times for 5 min each with reagent (4). After washing, the electrophoregrams are immersed in 0.5 N HCl for 2 min and placed in reagent (5) for 20 min. Finally, they are washed in acetone for 2 min and air-dried.

GLYCOPROTEIN STAINING ACCORDING TO SÜDHOF [27]

Reagents. 1) Dissolve 1.0 g of periodic acid in 30 ml of distilled water and add 70 ml of absolute ethanol.

2) Dissolve 1.0 g of parafuchsin in 30 ml of ice-cold 1 N HCl, add 1 g of potassium metabisulfite dissolved in 170 ml of distilled water while shaking, and leave the mixture for 48 h. Then, shake the solution with powdered charcoal and filter.

The parafuchsin used must not contain acridine. Potassium metabisulfite prepared long ago should not be used. If the container seal is broken, the top layer of the reagent should be discarded.

3) Formaldehyde-ammonia mixture solution. Mix equal volumes of 1 M formaldehyde and 1 N ammonium hydroxide.

4) Differentiating solutions: a) 96% ethanol and b) ethanol – 1 N HCl mixture (5:3).

5) Diethyl ether.

Procedure. Electrophoregrams are treated with reagent (1) for 10 min, then washed with running tap water for 10 min and immersed in distilled water for 2 min. After that, they are stained with reagent (2) until red glycoprotein zones appear against a colorless background. As soon as the background of the electrophoregram begins to turn pink, the strip should be quickly transferred to reagent (3), where it will turn completely red. After about 1–3 min, when the staining reaches maximum intensity, the electrophoregrams are immersed in reagent (4a) for 5 min, and then in reagent (4b), where a color change occurs: glycoproteins become reddish-purple, and the background is decolorized. Complete decolorization of the background is achieved by several changes of reagent (4b). After that, to remove HCl, the electrophoregrams are washed again with reagent (4a). Finally, they are immersed in ether and air-dried.