Experimental method for separation of serum proteins by cellulose acetate membrane electrophoresis
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September 17 05:00:30, 2025
**Experimental Principle**
Electrophoresis is a technique used to separate charged molecules, such as proteins, based on their migration in an electric field. When a protein carries a net charge, it moves toward the electrode with the opposite charge. In serum, most proteins have isoelectric points below pH 7, meaning they become negatively charged in a buffer solution at pH 8.6. Under these conditions, all serum proteins migrate toward the anode. However, due to differences in charge and molecular size, each protein migrates at a different speed. Smaller, more highly charged proteins move faster, while larger or less charged ones move slower. This allows the separation of serum proteins into five distinct fractions: albumin, α1-globulin, α2-globulin, β-globulin, and γ-globulin. After staining, the relative amounts of each fraction can be determined.
Acetate fiber membrane (CAM) is commonly used as a support medium for electrophoresis. After separation, the membrane is stained to visualize the protein bands. The intensity of the stain is proportional to the amount of protein present. Each band can be cut out and eluted in a dilute NaOH solution for colorimetric analysis. Alternatively, the membrane can be dissolved in an organic solvent and scanned using a densitometer for quantitative results.
This method offers several advantages, including high resolution, rapid processing, and minimal sample requirements. It is widely applied in clinical settings for analyzing serum proteins, hemoglobin, lipoproteins, and isoenzymes.
**Experimental Reagents**
1. Barbital-Barbital sodium buffer (pH 8.6, 0.06 M): Dissolve 2.21 g of barbital and 12.36 g of barbital sodium in 500 mL of distilled water, heat to dissolve, and dilute to 1 L.
2. Staining solutions:
- Li Chunhong S: 0.4 g Li Chunhong S + 6 g trichloroacetic acid in 100 mL distilled water.
- Amino Black 10B: 0.1 g amino black 10B in 20 mL absolute ethanol, add 5 mL glacial acetic acid, then mix with 2.5 g sulfosalicylic acid in 100 mL water.
3. Rinsing solutions:
- 3% acetic acid for Ponceau S.
- 45 mL methanol, 5 mL glacial acetic acid, and 50 mL water for Amino Black 10B.
4. Transparent liquid: 75 mL absolute ethanol + 25 mL glacial acetic acid.
5. Eluents:
- 0.1 mol/L NaOH for Ponceau S.
- 0.4 mol/L NaOH for Amino Black 10B.
6. 40% acetic acid solution.
**Equipment and Materials**
- Equipment: Electrophoresis apparatus, electrophoresis tank, spectrophotometer or densitometer.
- Materials: Acetate film, petri dish, filter paper, tweezers, spotter, ruler, pencil, scissors, etc.
**Procedure**
1. Prepare the electrophoresis tank by filling both sides with buffer, ensuring equal levels. Place filter paper between the two sides to connect the CAM.
2. Cut the acetate film (2 cm × 8 cm), mark a line 1.5 cm from one end, and soak the matte side in buffer for 20–30 minutes. Remove excess buffer with filter paper.
3. Spot 3–5 μL of non-hemolyzed serum at the marked line. Ensure even application and avoid damaging the film.
4. Balance the film by placing it in the tank, aligning the sample end with the cathode. Allow 5 minutes before starting electrophoresis.
5. Apply 10–15 V/cm voltage and 0.4–0.6 mA/cm current for 40–60 minutes, depending on season. Stop when the migration front reaches 3.5–4 cm.
6. Immediately immerse the film in the appropriate staining solution (Ponceau S or Amino Black 10B) for 5–10 minutes.
7. Rinse the film in sequential rinsing solutions until background is clear.
8. Quantify:
- For Amino Black 10B: Elute in 0.4 mol/L NaOH, measure absorbance at 620 nm.
- For Ponceau S: Elute in 0.1 mol/L NaOH, neutralize with acetic acid, measure at 520 nm.
- Alternatively, transparent the film and scan with a densitometer at 520 nm.
9. Calculate percentages using absorbance values.
10. Clinical interpretation: Abnormal patterns may indicate liver disease, kidney dysfunction, or multiple myeloma.
**Precautions**
- Avoid touching the buffer or CAM during power operation.
- Exchange electrodes regularly to maintain buffer pH.
- Keep buffer levels consistent to prevent electroosmosis or siphoning.
- Common issues include poor separation due to over-spotting, uneven migration, or improper film preparation.
- Dye choice affects accuracy; Ponceau S provides better resolution than Amino Black 10B.
- Fresh samples are essential; hemolysis can falsely elevate β-globulin.
This detailed procedure ensures accurate and reliable results, making it a valuable tool in biochemical and clinical analysis.