Serum protein agarose gel electrophoresis can be applied to (1) determine the protein content of serum and (2) diagnose diseases.
Operation method
Serum Protein Agarose Gel Electrophoresis
Principle
Agarose forms a gel mainly through hydrogen bonding. During electrophoresis, because of the large water content of the gel (98-99%), it is close to free electrophoresis, and because of the little influence of solid support, the electrophoresis speed is fast and the zones are neat. And because agarose does not contain charged groups, the influence of electroosmosis is very little, it is a better electrophoresis material, and the separation effect is better. Lipids in serum are combined with serum apolipoproteins to form water-soluble lipoproteins. The types and number of apolipoproteins contained in various lipoproteins are different, and the sizes of various lipoprotein particles are also very different. Therefore, agarose gel is used as a support, and various lipoprotein particles can be separated in the electric field. The method of separating serum proteins by agarose gel electrophoresis is simple. Serum lipoproteins are pre-stained with the lipid dye Sudan black (or oil red, etc.). Then the pre-stained serum is spiked in the agarose gel plate spiking tank, and after electrification, the lipoproteins can be seen to move toward the positive pole, and several bands are separated.
Materials and Instruments
Serum Samples Move 1, pre-staining serum serum 0.2 ml plus Sudan black staining solution 0.2 ml, mixed in a 37 ℃ water bath staining 30 minutes, centrifugation (2000 rpm) about 5 minutes. Centrifuge (2000 rpm) for about 5 minutes to remove the dye sediment suspended in the serum. 2、Preparation of agarose gel plate will have been prepared 0.5% agarose gel in a boiling water bath heating and melting, with a pipette pipetting gel solution poured on the slide, about 3 ml. half an hour of solidification (hot days need to be prolonged, but also can be placed in the refrigerator for a few minutes to accelerate the solidification). 3, point sample will be cut filter paper strip folded in half, with the folded edge at the end of the gel 2 cm from the cut point sample opening. Insert the capillary tube into the pre-stained serum, and after inhaling part of the serum, take the capillary tube so that the end with the sample is leaning against the end of the sampling port, and stop for about 3 seconds. 4、Electrophoresis put the gel plate flat into the electrophoresis tank, so that the sample end is connected to the cathode side, with four layers of filter paper or gauze to make a "bridge", laid on the two ends of the gel plate, each resting on the gel plate for about 1cm, the other end of the "bridge" immersed in the electrophoresis tank in the barbiturate buffer. The other end of the "bridge" was immersed in the barbiturate 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, then the separated zones can be observed. 5、If you need to keep the electrophoretic pattern, you can put the gel plate (together with the slide) after electrophoresis into water to soak and desalinate for 2 hours, and then put it into the oven (about 80℃) to dry. Caveat 1、Electrophoresis samples should be fresh fasting serum. 2、When heating and dissolving agar, it is necessary to prevent too much water evaporation. It is better to make agarose gel as soon as it is used so as not to dry the surface of the gel and affect the separation effect. 3， the production of gel plate agarose concentration generally choose 0.5% or so appropriate, higher than 1% above the α-lipoprotein part of the tighter, β and β-lipoprotein part of the former β-lipoprotein is not clear enough; lower than 4.5% of the coagulability is poor, the map is not clear. 4， the sampling port should be of appropriate size, with neat and smooth edges, otherwise it will affect the electrophoresis pattern. 5、If there is a shallow zone in front of α-lipoprotein, it can be listed as former α-lipoprotein. Common Problems Agarose is made from agar, which is selected for its purer texture. Agar is chemically a complex of agarose and agar gum. Agar gum is a polysaccharide containing sulfate and hydroxyl groups, which have ion-exchange properties that adversely affect electrophoresis and gel filtration. Agarose is a straight chain polysaccharide which consists of alternating residues of D-galactose and 3,6-anhydro-L-galactose. For more product details, please visit Aladdin Scientific website.
Sudan black staining solution Anhydrous ethanol Barbiturate buffer Gel buffer Agarose gel
Water Bath Electrophoresis Instrument Electrophoresis Tank Centrifuge