Protein technology topic: serum protein agarose gel Electrophoresis
Agarose is made by selecting pure AGAR as raw material. Agarose is chemically a complex of agarose and agarose gum. Agar-gel is a polysaccharide containing sulfate and hydroxyl groups. It has ion-exchange properties, which affect electrophoresis and gel filtration. Agarose is a straight-chain polysaccharide consisting of an alternate arrangement of d-galactose and 3, 6-dehydration-L-galactose residues.
Agarose forms a gel mainly by hydrogen bonding. During electrophoresis, because of the large water content of gel (98-99%), it is nearly free electrophoresis. Because the influence of solid support is less, the electrophoresis speed is fast and the zone is neat. Moreover, since agarose does not contain charged groups and has little electroosmotic effect, it is a better electrophoretic material with better separation effect.
Serum lipids are present in the form of water-soluble lipoproteins that bind to serum apolipoproteins. The type and quantity of apolipoprotein in various lipoproteins are different, and the size of lipoprotein particles is also very different. Therefore, with agarose gel as the support, various lipoprotein particles can be separated in the electric field.
The separation of serum protein by agarose gel electrophoresis is simple. The serum lipoproteins were pre-dyed with the lipid dye Sudan black (or oil red, etc.). The pre-dyed serum was then added to the agarose gel plate sampling tank, and the lipoprotein could be seen to move towards the positive pole and separate out several zones after electrification.
In normal serum lipoprotein, there are three zones, from cathode to anode, β -lipoprotein (deep), pre-β -lipoprotein (shallow) and α -lipoprotein (slightly deeper than pre-β -lipoprotein), and there should be no chylomicrons at the origin. Sometimes the pre - β -lipoprotein doesn't show up.
Agarose forms a gel mainly by hydrogen bonding. During electrophoresis, because of the large water content of gel (98-99%), it is ne
1. Pre-stained serum 0.2mL of serum was mixed with 0.2ml of Sudan black staining solution, and then placed in a water bath at 37℃ for dyeing for 30 min, centrifuged at 2000 RPM for about 5 min. To remove dye deposits suspended in serum.
2. Preparation of agarose gel plate The prepared 0.5% agarose gel was heated and melted in a boiling water bath, and the gel solution was absorbed with a pipette and poured on the slide, about 3 ml. Leave to set for half an hour (longer on hot days, or refrigerate for a few minutes to speed up setting).
3. Spot samples fold the cut filter paper strip in half and cut a sample 2cm away from one end of the gel with hem. Insert the capillary into the pre-dyed serum. After some of the serum has been inhaled, take the capillary with the end of the sample resting against the tip of the sampling port for about 3 seconds.
4. Electrophoresis put the gel plate flat in the electrophoresis tank, and make the sample addition end connected to one side of the cathode. Use four layers of filter paper or gauze to make "approach bridge" and apply it to both ends of the rubber plate, each of which holds the gel plate about 1cm, and the other end of the "approach bridge" is immersed in the buffer solution in the electrophoresis tank. Turn on the power, first adjust the current to 3-4mA/ gel plate, electrophoresis for 10-15 minutes; Then the current was adjusted to 6-7mA/ gel plate and electrophoresis was performed for 30-40 minutes to observe the separated bands.
5. If you need to retain the electrophoretic map, you can put the gel plate after electrophoresis (together with the slide) in clear water for soaking and desalting for 2 hours, and then put it in the oven (about 80℃) to dry. "Reagent"
1. Sudan black stain solution Sudan black B is added to anhydrous ethanol until it is saturated and shaken to acetyl. Filter before use.
2. Buffer solution 15.4g sodium, 2.76g and 0.29g EDTA were weighed and dissolved with water, and then distilled water was added to 1000mL (pH 8.6, ionic strength 0.075) to form the electrode buffer solution.
3. Take 1.212g of trihydroxymethyl aminomethane, 0.29g of EDTA and 5.85g of nacL as gel buffer, dissolve them in distilled water, and then dilute them to 1000mL with pH of 8.6.
Agarose gel 0.45g of agarose was weighed and dissolved in 50 ml of gel buffer, and 50 ml of water was added. Heat in a water bath until boiling, and stop heating immediately after the agarose is completely dissolved.
[Matters needing attention]
1. The electrophoretic sample should be fresh fasting serum.
2. When heating and melting AGAR, it is necessary to prevent excessive evaporation of water. Agarose gel was prepared as needed to avoid drying the surface of gel and affecting the separation effect.
3, the production of gel plate agarose concentration is generally selected about 0.5% is appropriate, higher than 1% α -lipoprotein part is closer, β and pre-β -lipoprotein part is not clear; Less than 4.5% of the solidification is poor, the map is unclear.
4. The sample mouth should be suitable in size, and the edge should be neat and smooth, otherwise it will affect the electrophoretic map.
5. If there is a shallow zone in front of α -lipoprotein, it can be classified as anterior α -lipoprotein.
[Normal reference range]
α -lipoprotein 20~30%
β -lipoprotein 20~30%
Pre-β -lipoprotein 0 to 28%