Key Points of Electrophoresis Operation Using Cellulose Acetate Membrane and Common Errors Analysis
Cellulose acetate is a derivative of cellulose, formed by the acetylation of hydroxyl groups in cellulose. A thin film made from this material is known as a cellulose acetate membrane. This membrane has minimal protein adsorption, effectively eliminating the "tailing" effect commonly seen in paper electrophoresis. Due to its low hydrophilicity, it contains less buffer, allowing most of the current to be carried by the sample during electrophoresis. As a result, the separation speed is fast, the electrophoresis time is short, and only small amounts of sample are needed—such as 5 μg of protein—to achieve good separation. This makes it ideal for detecting trace abnormal proteins in pathological conditions.
Compared to filter paper, cellulose acetate membranes offer several advantages:
(1) Excellent separation performance. The membrane shows very little adsorption of proteins, no "tailing," and clear banding after staining with no background interference, which improves the accuracy of quantitative analysis.
(2) Fast and efficient. Because of its lower hydrophilicity, the electroosmotic flow is minimized, and most of the current is conducted through the sample. This results in faster separation, typically taking 45–60 minutes, plus staining and decolorization, completing the entire process in about 90 minutes.
(3) High sensitivity and low sample volume. Only 2 μL of serum is required for serum protein electrophoresis, making it suitable for detecting subtle changes in abnormal proteins under disease conditions.
(4) Broad application range. Some proteins that are difficult to separate on paper, such as fetal gamma globulin, lysozyme, insulin, and histone, can be effectively separated using cellulose acetate membranes.
(5) Easy storage and quantification. After staining, the membrane can be made into a transparent dry plate by immersing it in a glacial acetic acid-ethanol mixture or other transparent solutions, facilitating scanning and long-term preservation.
To make the membrane transparent for light absorption scanning and long-term storage, it is treated with a solution of glacial acetic acid and ethanol or other similar liquids.
1. Materials and Reagents
Commercially available cellulose acetate membranes are typically used. The most common electrophoresis buffer is barbital buffer at pH 8.6, with a concentration ranging from 0.05 to 0.09 mol/L.
2. Operating Procedures
(1) Membrane Pretreatment: Before electrophoresis, the membrane must be soaked in the buffer solution. After soaking, remove excess buffer using filter paper and mark the corner.
(2) Sample Loading: The amount of sample depends on factors such as sample concentration, nature, staining method, and detection technique. For routine serum protein electrophoresis, the sample line should not exceed 1 μL per cm, equivalent to 60–80 μg of protein.
(3) Electrophoresis: Conducted at room temperature, with a voltage of 25 V/cm and a current of 0.4–0.6 mA/cm width.
(4) Staining: Amino black and Ponceau S are commonly used for general protein staining. Toluidine blue or periodic acid-Schiff reagent is used for glycoproteins, while Sudan black or magenta sulfite is used for lipoproteins.
(5) Decolorization and Transparency: A 5% acetic acid solution is commonly used for water-soluble dyes. For long-term storage or scanning, the membrane can be immersed in a 30:70 (V/V) mixture of glacial acetic acid and absolute ethanol.
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