Cellulose acetate membrane electrophoresis is an electrophoresis method that uses cellulose acetate membrane as a support. It is acetate of cellulose, produced by acetylation of the hydroxyl groups of cellulose. It is dissolved in organic solutions such as acetone and can be coated into a uniform and fine microporous film with a thickness of 0.1-0.15 mm.
In this experiment, cellulose acetate membrane electrophoresis was used to separate serum proteins. The method is to use a spotter to spot a small amount of fresh serum on a cellulose acetate membrane soaked in buffer. Both ends of the membrane were connected to the buffer in the electrophoresis tank through filter paper, and the pH of the buffer used was 8.6. Serum proteins are negatively charged in this buffer and move towards the positive electrode in the electric field. Different proteins in serum have different swimming speeds due to different charges and relative molecular masses. Those with more charge and smaller relative molecular mass swim faster, while those with less charge and larger relative molecular mass swim slower. After a certain period of time, take out the membrane and immediately immerse it in the staining solution to fix and stain the protein. Then move the membrane into the immersion solution and wash until the background is colorless. At this time, blue bands appear on the membrane, each band representing a protein. According to the order of swimming speed, each zone is albumin, α1-globulin, α2-globulin, β-globulin and γ-globulin.
Cellulose acetate film has been widely used because it has no adsorption to the sample, clear boundaries between zones during electrophoresis, no obvious tailing, small amount of sample, and short electrophoresis time.
Cut the membrane into 2.5 cm × 8 cm pieces. Draw a line with a pencil about 2 cm from one end of the matte surface (front) of the membrane to mark the spotting position.
Use tweezers to place the matte side of the membrane downward and float it on the surface of the barbiturate buffer (the buffer is in a petri dish), allowing the membrane strip to soak and sink naturally. About 10 min.
Take out the membrane and use filter paper to absorb the surface water. Then lay it flat on the slide (matte side facing up), dip the cover slip in the serum in the small petri dish, then gently drop it horizontally 2 cm away from one end of the membrane strip and then lift it up. In this way, a thin strip of serum sample is spotted on the membrane strip.
Add buffer solution to the electrophoresis tank to make the liquid levels in the two electrode tanks equal, and suspend the membrane strip flat on the filter paper bridge of the electrophoresis tank bracket (first cut a filter paper strip of appropriate size, attach the double-layer filter paper strip to the bracket of the electrophoresis tank, align one end of it with the front edge of the bracket, and immerse the other end in the buffer solution of the electrode tank. Wet the filter paper completely with the buffer solution and drive out the bubbles, so that the filter paper is tightly attached to the bracket, which is the filter paper bridge). The spotting end is close to the negative electrode, balance for 10 min, turn on the power, adjust the current to 0.4-0.6 mA, the voltage to 10-12 V per centimeter, and perform electrophoresis for 45-60 min.
Take out the membrane strip and place it in the color developing solution for 10 min.
Rinse the membrane strip several times in the rinse solution until the background color is removed, and a clear electrophoresis map can be obtained.
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