ã€Reagents】
1. Sudan black staining solution
Sultan Black B was added to absolute ethanol to saturation and shaken to acetylate. Filter before use. Shanghai Chuangsai Technology provides 4197-25-5, Sudan Black B, GR, commodity number: D16-1032969-100G, the price is 431 yuan.
2, barbital buffer
Weighed 15.4 g of barbital sodium, 2.76 g of barbital and 0.29 g of EDTA. After adding water, distilled water was added to 1000 mL (pH 8.6, ionic strength 0.075) to prepare an electrode buffer. Shanghai Chuangsai Technology provides barbituric acid, Barbituric acid, 99.5%, for colorimetric analysis of cyanide derivatives and cyanide determination, 67-52-7, commodity number: C16-B10490-25g, price 290 yuan.
3, gel buffer
1.212 g of tris (hydroxymethyl)aminomethane, 0.29 g of EDTA, and 5.85 g of NaCl were dissolved in distilled water, and then diluted to 1000 mL, and the pH was 8.6. Shanghai Chuangsai Technology provides 77-86-1, Tris, Tris>99%, molecular biology, commodity number: C84-3739-100g, price 65 yuan.
4, agarose gel
Weigh 0.45 g of agarose in 50 mL of gel buffer and add 50 mL of water. Heat to boiling in a water bath and stop heating immediately after the agarose is completely dissolved.
ã€operating】
1, pre-stained serum
0.2 mL of Sudan black staining solution was added to 0.2 mL of serum, mixed and stained in a 37 ° C water bath for 30 minutes, and centrifuged (2000 rpm) for about 5 minutes. To remove the dye residue suspended in the serum.
2. Preparation of agarose gel plate
The prepared 0.5% agarose gel was heated and thawed in a boiling water bath, and the gel solution was pipetted onto a glass slide, about 3 mL. Allow to stand for half an hour after solidification (expanded when hot, or put into the refrigerator for a few minutes to accelerate coagulation).
3, spotting
Cut the cut filter paper in half and cut a bit at 2 cm from the end of the gel with the fold. Insert the capillary into the pre-stained serum. After inhaling part of the serum, take the capillary so that one end of the sample is placed against the end of the sample mouth and stop for about 3 seconds.
4, electrophoresis
Place the gel plate in the electrophoresis tank, and connect the sample to the cathode side. Make a “bridge†with four layers of filter paper or gauze, and apply it to both ends of the rubber plate. Each gel plate is about 1cm. The other end of the "bridge" is immersed in the barbital buffer in the electrophoresis tank. Turn on the power, first adjust the current to 3-4mA / gel plate, electrophoresis for 10-15 minutes; then adjust the current to 6-7mA / gel plate, electrophoresis for 30-40 minutes, you can observe the separation zone.
5, retain the electropherogram
The gel plate after electrophoresis (along with the slide) can be immersed in clear water for 2 hours, and then dried in an oven (about 80 ° C).
ã€Precautions】
1. The electrophoresis sample should be fresh fasting serum.
2. When heating and melting agar, it is necessary to prevent excessive evaporation of water. The agarose gel is preferably used as needed to prevent the gel surface from drying and affecting the separation effect.
3. When preparing the gel plate, the agarose concentration is generally about 0.5%, the α-lipoprotein fraction is more than 1%, the β and pre-beta-lipoprotein fractions are not clear enough; the coagulation is less than 4.5%. Poor, the map is unclear.
4, the sample mouth should be the appropriate size, the edges are neat and smooth, otherwise it will affect the electropherogram.
5. If there is a shallow zone before α-lipoprotein, it can be listed as pre-alpha-lipoprotein.
[normal reference range]
--lipoprotein 20~30%
--lipoprotein 20~30%
Pre-β-lipoprotein 0~28%
Milk thistle particles (-)
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