After serum lipoproteins are stained with Sudan black B and electrophoresed in pH 8.6 barbiturate buffer with agarose, the lipoproteins can be divided into different zones. Depending on the speed of electrophoretic movement, normal human serum lipoproteins can appear in three bands, in order from cathode to anode, as β-lipoproteins (the darkest), pre-beta-lipoproteins (the lightest), and α-lipoproteins (slightly darker than pre-beta-lipoproteins). There should be no celiac particles at the origin, and no bands of intermediate lipoproteins are seen, and sometimes pre-beta-lipoproteins are not shown.
Operation method
Serum lipoprotein agarose electrophoresis assay
Principle
After serum lipoproteins are stained with Sudan black B and electrophoresed in pH 8.6 barbiturate buffer with agarose, the lipoproteins can be divided into different zones. According to the different speeds of electrophoretic movement, normal human serum lipoproteins can appear in three bands, from cathode to anode, β-lipoproteins (the darkest), pre-beta-lipoproteins (the lightest), and α-lipoproteins (slightly darker than pre-beta-lipoproteins). There should be no celiac particles at the origin, and no bands of intermediate lipoproteins are seen, and sometimes pre-beta-lipoproteins are not shown. If the pre-beta-lipoprotein is darker than the alpha-lipoprotein and serum triglycerides are markedly elevated and cholesterol is normal or slightly elevated, type IV hyperlipidemia can be identified. beta-lipoprotein bands that are markedly darker than normal and serum total cholesterol that is markedly elevated with normal triglycerides are considered to be type IIa hyperlipidemia, and those with elevated serum total cholesterol that is slightly elevated with high triglycerides and darker pre-beta are considered to be type IIb hyperlipidemia. beta-lipoprotein and pre-beta-lipoprotein are not clearly distinguishable from each other. The two bands are indistinguishable from each other as the "wide beta band", and if both serum triglycerides and cholesterol are increased, it can be classified as type III hyperlipidemia. The presence of celiac disease at the origin, normal or decreased β and pre-beta, and a marked increase in serum triglycerides can be classified as type I hyperlipidemia.
Materials and Instruments
Serum Move I. Reagents Caveat 1. Electrophoresis samples should be fresh fasting serum. 2. When heating and dissolving the agar, it is necessary to prevent too much water evaporation. The agar gel should be made as soon as it is used, so as to avoid drying of the gel surface, which may affect the separation results. 3. If there is a shallow zone in front of α-lipoprotein, it can be classified as α-pre-lipoprotein. 4. If you want to save the electrophoretic pattern, you can soak the gel plate (together with the slide) in water for 2 hours to desalinate, and then dry it in the drying oven (about 80℃). 5. The concentration of agarose used for making the gel plate is generally about 0.5%; if it is higher than 1%, the part of α-lipoprotein will be more compact, and the part of β- and pre-beta-lipoprotein will not be clear enough; if it is lower than 0.45%, the gel will be poorly coagulated, and the graphs will not be clear. 6. The sample tank should be of suitable size with neat and smooth edges, otherwise it will affect the electrophoresis pattern. For more product details, please visit Aladdin Scientific website.
Sudan Black B Stain Barbiturate Buffer Tris Buffer
Agarose gel Horizontal electrophoresis equipment Slides Centrifuge Water baths
1. serum.
2. Sudan black B staining solution Saturated anhydrous ethanol solution of Sudan black B, filtered before use.
3. Barbiturate buffer (pH 8.6, ionic strength 0.075) as electrode buffer. Sodium barbital 25.4g, barbital 2.76g, EDTA acid 0.292g, dissolved with water and diluted to 1000mL with water.
4. Tris buffer (pH 8.6) for gel buffer. tris 1.212g, EDTA acid 0.292g NaCL 5.85g, add water to dissolve and then add water to 1000mL.
5. agarose gel Agarose 0.45g, Tris buffer 50mL, add water 50mL, heating to boiling while stirring, stop heating immediately after the agarose is dissolved.
6. Horizontal electrophoresis equipment.
7.37℃ water bath.
8. Centrifuge.
9. Slides
10. incision knife blade length of 15mm two pieces of blade, the center of the clip a plexiglass or wood, fixed with screws, so that the two pieces of blade 1.5mm apart. with the corresponding groove better.
11. grooving small spoon with a diameter of 1.5mm copper wire about 6cm long, one end hammered into a flat, with fine sandpaper polished (with a good X-ray film can also be cut).
Hematocrit tube or microfuge.
Second, the operation steps
1. Pre-staining serum Serum 0.2mL plus Sudan Black B Staining Solution 0.02mL in a small test tube, mixed and placed in a 37℃ water bath for 30 minutes. Then centrifuge at 2000r/min for 5 minutes.
2. Preparation of agarose gel plate The 0.45% agarose gel that has been configured was placed in a boiling water bath and heated to melt. Pipette the gel solution onto the slides, about 2.5mL per slide, and let it stand for about half an hour to solidify (prolonged when it is hot, or put it in the refrigerator for a few minutes to accelerate the solidification).
3. Spot addition of serum At about 2 cm from one end of the solidified agarose gel plate, use an incision blade (groover) to cut vertically into the gel and remove it immediately, and then use a small copper wire spoon to remove the small rectangular strips of gel. Drain the water in the small slot with a small piece of filter paper, and aspirate about 15 μl of pre-stained serum with a heme pipette and inject it into the small slot on the gel plate.
4. Electrophoresis Place the serum-added gel plate parallel to the electrophoresis tank, with the sample placed at the cathode end. Wet two pieces of three-layer gauze in barbiturate buffer, and then gently and tightly attached to the ends of the gel plate, the other end of the gauze dipped in the barbiturate buffer in the electrophoresis tank (Note: this electrode buffer can not be replaced by trimethylolamine methane buffer). Turn on the power supply, the voltage is 120~130V, the current is 3~4mA for each piece. about 45 to 55 minutes, you can see the separated color band.