1. [principle]

  2. [operation]

  3. [reagent]

  4. [precautions]

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Key words: Agar electrophoresis serum

Keywords: Agar electrophoresis serum serum protein serumprotein agarose gel electrophoresis agarosegelelectrophoresis

[principle]

Agarose is selected and made from agar which is pure in texture. Agar is a complex of agarose and agarose. Agar gel is a polysaccharide containing sulfate and hydroxyl, which has ion exchange property. This property will have bad effects on electrophoretic and gel filtration. Agarose is a straight chain polysaccharide, which consists of D-galactose and 3,6-dehydrated-l-galactose residues arranged alternately.

Agarose is mainly formed by hydrogen bonding. Electrophoretic time is approximately free electrophoresis because of the large water content of the gel (98 - 99%). Because the influence of solid support is less, the electrophoresis speed is fast and the zone is neat. Moreover, agarose is a good electrophoretic material because it does not contain charged groups and has little effect on electroosmosis.

The lipids in serum combine with apolipoproteins to form water-soluble lipoproteins. The size and size of apolipoproteins in various lipoproteins are different. The sizes of various lipoproteins are also very different. Therefore, agarose gel can be used as a support to separate various lipoprotein particles in the electric field.

The method of sedating serum proteins by agarose gel electrophoresis is simple. The serum lipoproteins were pre dyed with Sudan black (or oil red, etc.). Then the prestained serum was added to the agarose gel plate plus sample tank. After electrified, lipoproteins could be seen to move to the positive electrode and several zones were isolated.

There are three bands in normal human serum lipoproteins. From the cathode to the anode, they are β - lipoproteins (the deepest), pre β - lipoproteins (the shallowest) and α - lipoproteins (slightly deeper than pre β - lipoproteins). There should be no chyle particles at the origin. Sometimes pre - β - lipoproteins also show

It doesn't show.

Agarose is mainly formed by hydrogen bonding. Electrophoretic time is approximately free electrophoresis because of the large water content of the gel (98 - 99%). Because the influence of solid support is less, the electrophoresis speed is fast and the zone is neat. Moreover, agarose is a good electrophoretic material because it does not contain charged groups and has little effect on electroosmosis.

The lipids in serum combine with apolipoproteins to form water-soluble lipoproteins. The size and size of apolipoproteins in various lipoproteins are different. The sizes of various lipoproteins are also very different. Therefore, agarose gel can be used as a support to separate various lipoprotein particles in the electric field.

The method of sedating serum proteins by agarose gel electrophoresis is simple. The serum lipoproteins were pre dyed with Sudan black (or oil red, etc.). Then the prestained serum was added to the agarose gel plate plus sample tank. After electrified, lipoproteins could be seen to move to the positive electrode and several zones were isolated.

There are three bands in normal human serum lipoproteins. From the cathode to the anode, they are β - lipoproteins (the deepest), pre β - lipoproteins (the shallowest) and α - lipoproteins (slightly deeper than pre β - lipoproteins). There should be no chyle particles at the origin. Sometimes pre - β - lipoproteins are not shown.

[operation]

  1. 0.2ml of Sudan black staining solution was added to 0.2ml of pre dyed serum, mixed in a 37 ℃ water bath for 30 minutes, and centrifuged (2000 rpm) for about 5 minutes. To remove dye sediment suspended in serum.

  2. Agarose gel plate was prepared and heated by boiling the 0.5% agarose gel prepared in the boiling water bath. The straw was sucking on the glass slide by suction tube and about 3 mL. Allow to stand for half an hour before setting (it needs to be prolonged when it is hot, or put it in the refrigerator for several minutes to accelerate the setting).

3, the sample will fold the cut filter paper, fold the edge and cut a bit of 2cm at the end of the gel. Insert the capillary into the pre dyed serum, take the capillary to make the end with sample lean against the end of the sample port, and stop for about 3 seconds.

4, electrophoresis will put the gel plate into the electrophoresis tank, so that the sample end will be connected to the cathode side, and use four layers of filter paper or gauze to make the "approach bridge". It will be applied to the two ends of the rubber plate, and the gel board will be around 1cm. The other end of the approach bridge will be immersed in the barbiturate buffer solution in the electrophoresis tank. To switch 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, we can observe the separated zone.

5, if the electrophoresis pattern is needed, the gel plate (together with the slide) can be put into the clear water for 2 hours and then dried in the oven (about 80 degrees Celsius).

[reagent]

  1. Sudan black dye solution add Sudan black B to anhydrous ethanol until it is saturated, and shake to acetylate. Filter before use.

  2. Barbital buffer solution: weigh 15.4g of barbital sodium, 2.76g of barbital and 0.29g of EDTA, add distilled water to 1000ml (pH: 8.6, ionic strength: 0.075) after water dissolving, which is the electrode buffer solution.

3, gel buffer solution takes three hydroxymethyl amino methane 1.212g, EDTA0.29g and NaCl5.85g, dissolves with distilled water, dilute to 1000mL, pH is 8.6.

  1. Agarose gel was used to extract agarose 0.45 g dissolved in 50 mL gel buffer and add 50 mL of water. Heat to boiling in water bath, stop heating immediately after agarose is completely dissolved.

[precautions]

  1. The electrophoresis sample should be fresh fasting serum.

  2. When heating the dissolved agar, it is necessary to prevent excessive evaporation of water. Agarose gel is best used with the gel so as to prevent the gel surface from drying and affect the separation effect.

3, when making gel plates, agarose concentration is generally about 0.5%, which is higher than 1%, and the proportion of alpha lipoprotein is relatively tight. The part of beta and pre beta lipoprotein is not clear enough. Below 4.5%, the coagulability is poor and the atlas is not clear.

  1. The size of the sample port should be suitable, and the edge should be neat and smooth, otherwise the electrophoretic pattern will be affected.

  2. If there is a shallow zone before α - lipoproteins, it can be classified as pre α - lipoproteins.

[normal reference range]

α - lipoproteins 20-30%

β - lipoproteins 20-30%

Pre β - lipoproteins 0-28%

Chylomicrons (-)

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