Experiment for separating serum protein by Cellulose Acetate Membrane Electrophoresis

Principle

Cellulose acetate film electrophoresis is an electrophoresis method using a cellulose acetate film as a support. The phenomenon in which charged particles move toward the opposite electrode under the action of electric field is called electrophoresis. Since each protein has a specific isoelectric point, if a protein is placed in a solution with a pH lower than its isoelectric point, the protein will be positively charged and move toward the negative electrode. On the contrary, it moves to the positive pole. Because the speed of protein molecules moving in an electric field is related to their charge, the shape and size of the molecules, different proteins can be separated by electrophoresis. Serum contains a variety of proteins, all of which have isoelectric points at pH7.5 or less. When serum is placed in pH 8.6 buffer to run electrophoresis, all serum proteins are negatively charged and will move towards the positive side in the electric field. Since various serum proteins have different charges at the same pH, the molecular particles are different in size, and thus the migration speed is different, and they are separated by electrophoresis. The isoelectric points and molecular weights of serum proteins are shown in the table below:

Protein

 Isoelectric points(PI)

Molecular weight

Albumin

4.88

69 000

α1-gloulin

5.00

200 000

α2-gloulin

5.06

300 000

β-gloulin

5.12

9 000~150 000

γ-gloulin

6.85~7.50

156 000~ 300 000

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The experiment is to separate different proteins in blood serum with cellulose acetate membrane (abbr. CAM) as support media. CAM is a kind of foamed loose film with good uniform, and the thickness is 0.1mm-1.5mm, which has a certain water absorption.

Materials, instruments and reagents for CAM Electrophoresis

Samples: healthy human blood serum

Instrument: power supply DYY-6C, electrophoresis tank DYCP-38C, superior sample loading WD-9404

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The reagents

1) cellulose acetate membrane 7X9cm

2) PH 8.6 barbitol buffer solution (ionic strength 0.05-0.09, it is 0.06tid time.): take 1,84g Diethyl barbituric acid, and then take 1.03g Diethyl sodium pentobarbital, add some distilled water to heat to dissolve, and make up to 1000ml ;

3) Stain: Ponceau S 0.2g, Trichloroacetic 3g, add 100ml distilled water;

4) TBS/T or PBS/T: 45ml 95% ethanol, 5ml glacial acetic acid, add 50ml distilled water;

5) Cleaning Solution: 70ml anhydrous ethanol, 30ml glacial acetic acid.

Experiment Method

1) Prepare the membrane: Immerse the membrane into the barbitol buffer solution 30min-8h, and take it out, remove the extra solution by absorbent paper.

2) Loading samples: Distinguish the rough side and smooth side of the membrane, and mark a line at 1.5cm distance to the top end on the rough side. Load samples by superior sample loading tool on the rough side. Note: The samples should be loaded on the rough side of membrane. After the serum samples are fully penetrated into membrane, turn over the membrane, put the rough side (with samples) face down to the tank, and the end with samples is placed in negative electrode.

3) Electrophoresis: Turn on the power supply, 0.40.6m A/cm membrane, the running time is 30-45 min. After running electrophoresis, turn off the power.

4) Stain and clean: Take out the membrane from the tank, and immerse it  into the dye solution for 5 min, and then clean it in the cleaning solution over and over for 3-4 times until the background color is clear. The serum proteins should be showed on the bands, and normally there are five zones, form top end to the marked line, Albumin, α1-gloulin, α2-gloulin, β-gloulin, γ-gloulin.

5) Preservation: put the dry elecrtophoresis band in cleaning solution for 10-15 minutes, and then take it out and stick it on a clean glass, after it drys, it will become a transparent film band.

Experiment Result

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The separation effect of serum samples is good, and there is no band tailing phenomenon. The repeatability of the results varies due to the test procedures and the methods of the experimenter, and the repeatability is high.

Conclusion

The rapid clinical electrophoresis detection system ( Electrophoresis tank DYCP-38C, power supply DYY-6C and superior sample loading WD-9404) produced by our company Beijing Liuyi Biotechnology Co., Ltd meets the experimental requirements, and the results are reproducible, simple and fast, suitable for teaching research.

Beijing Liuyi Biotechnology Co.,Ltd specializes in manufacturing elecrtophoresis products for life science industry, which has more than 50-year history in China and the company can provide stable and high-quality products all around the world. Through years of development, it is worthy of your choice!

We are now looking for partners, both OEM electrophoresis tank and distributors are welcomed.

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Post time: Nov-14-2022