Key Points of Electrophoresis Using Cellulose Acetate Membrane and Common Errors Analysis
Cellulose acetate is a derivative of cellulose formed by the acetylation of hydroxyl groups. A thin film made from this material is known as a cellulose acetate membrane. This membrane exhibits minimal protein adsorption, effectively eliminating the "tailing" effect commonly seen in paper electrophoresis. Due to its low hydrophilicity, it contains less buffer solution, allowing most of the current to pass through the sample during electrophoresis. As a result, the separation process is faster, the electrophoresis time is shorter, and only small amounts of sample are needed—approximately 5 μg of protein can yield excellent separation. This makes it particularly suitable for detecting trace abnormal proteins under pathological conditions.
Compared to filter paper, cellulose acetate membranes offer several advantages:
(1) Excellent separation quality. The membrane has minimal interaction with protein samples, preventing the "tailing" phenomenon. After staining, the background can be completely decolored, resulting in clear protein bands that enhance the accuracy of quantitative analysis.
(2) Fast and efficient. With lower hydrophilicity and reduced electroosmotic flow, most of the current is carried by the sample, leading to quicker separation. Typical electrophoresis times range from 45 to 60 minutes, and when including staining, decolorization, and scanning, the entire process can be completed within about 90 minutes.
(3) High sensitivity and low sample volume. For serum protein electrophoresis, only 2 μL of serum is required, making it ideal for detecting subtle changes in abnormal proteins in clinical settings.
(4) Wide applicability. Some proteins that are difficult to separate on paper, such as fetal gamma globulin, lysozyme, insulin, and histone, can be successfully separated using cellulose acetate membranes.
(5) Easy storage and quantification. After staining, the membrane can be made into a transparent dry plate by soaking it in a mixture of glacial acetic acid and ethanol, facilitating scanning and long-term preservation.
The cellulose acetate membrane is treated with a transparent solution like glacial acetic acid and ethanol to make it optically clear, which is beneficial for light absorption scanning and long-term storage.
**Materials and Reagents**
Commercially available cellulose acetate membranes are typically used. The most common electrophoresis buffer is a barbital buffer at pH 8.6, with concentrations ranging from 0.05 to 0.09 mol/L.
**Operating Procedures**
(1) Membrane Pretreatment: The membrane must be soaked in the buffer before electrophoresis. After soaking, excess buffer is removed using filter paper, and the corner is marked to indicate orientation.
(2) Sample Loading: The amount of sample depends on factors such as concentration, nature, staining method, and detection technique. For routine serum protein electrophoresis, the sample load should not exceed 1 μL per cm, equivalent to approximately 60–80 μg of protein.
(3) Electrophoresis: Conducted at room temperature. The voltage is set at 25 V/cm, and the current ranges between 0.4 to 0.6 mA/cm width.
(4) Staining: Amino black and Ponceau S are commonly used for general protein staining, while toluidine blue or periodic acid-Schiff reagent is used for glycoproteins. Sudan black or magenta sulfite is suitable for lipoprotein staining.
(5) Decolorization and Transparency: A 5% acetic acid solution is typically 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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