Cellulose acetate is a cellulose acetate formed by cellulose hydroxyacetylation. A film made of this material is called a cellulose acetate film. The film has little adsorption to the protein sample, and can almost completely eliminate the "tailing" phenomenon in paper electrophoresis. Because the membrane is less hydrophilic, it contains less buffer, and most of the current during electrophoresis is Sample conduction, so the separation speed is fast, the electrophoresis time is short, the sample dosage is small, and 5 μg of protein can obtain satisfactory separation effect. Therefore, it is particularly suitable for the detection of trace abnormal proteins under pathological conditions.
Compared with filter paper, cellulose acetate film has the following advantages:
(1) The separation effect is good. There is very little adsorption on protein samples, no “tailing†phenomenon, the background can be completely decolored after dyeing, and the separation of various protein dyeing bands is clear, thus improving the accuracy of quantitative determination.
(2) Fast and time saving. Because the hydrophilicity is smaller than the filter paper, the electroosmotic effect is small, and most of the current is transmitted by the sample during electrophoresis, so the separation speed is fast, the electrophoresis time is short, and the electrophoresis is generally 45 to 60 minutes, plus dyeing, decolorization, and whole electrophoresis. It takes only about 90 minutes to complete.
(3) High sensitivity, low sample dosage, serum protein electrophoresis requires only 2 μl of serum, so it is often used to detect trace abnormal protein changes under pathological conditions.
(4) Wide application. Some proteins are not easily separated by electrophoresis on paper. For example, fetal gamma globulin, lysozyme, insulin, histone, etc. can be separated by electrophoresis using cellulose acetate membrane.
(5) Easy to save, easy to quantify. After electrophoresis dyeing of cellulose acetate film, it can be made into transparent dry plate after being immersed in glacial acetic acid, ethanol mixture or other solution, which is beneficial to scanning quantitative and long-term preservation.
The cellulose acetate film is treated with glacial acetic acid ethanol solution or other transparent liquid to make the film transparent, which is beneficial to the light absorption scanning measurement of the electrophoresis pattern and the long-term preservation of the film.
1. Materials and reagents The cellulose acetate membrane is generally used commercially. The commonly used electrophoresis buffer is a barbital buffer of pH 8.6, and the concentration is 0.05-0.09 mol/L.
2. Operating points
(1) Pretreatment of the membrane: The membrane must be immersed in the buffer before electrophoresis. After soaking, the membrane is taken out and the excess buffer is removed by the filter paper, and the membrane is not absorbed. And mark the corner.
(2) Loading: The dosage of the sample is determined by factors such as the concentration of the sample, its nature, the dyeing method and the detection method. For routine electrophoresis analysis of serum proteins, the sample line per cm does not exceed 1 μl, which is equivalent to 60-80 μg of protein.
(3) Electrophoresis: It can be carried out at room temperature. The voltage is 25 V/cm and the current is 0.4 to 0.6 mA/cm wide.
(4) Dyeing: Amino black and Ponceau are commonly used for general protein staining, toluidine blue or periodic acid-Schiff reagent for glycoprotein, and lipoprotein is stained with Sudan black or magenta sulfite.
(5) Decolorization and transparency: The most commonly used decolorizing agent for water-soluble dyes is a 5% aqueous solution of acetic acid. For long-term storage or light absorption scanning measurement, it can be immersed in a transparent liquid of glacial acetic acid: absolute ethanol = 30:70 (V/V).
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