1. Install sandwich type vertical plate electrophoresis tank
Sandwich type vertical plate electrophoresis tank is easy to operate and not easy to leak. This electrophoresis tank is made of organic glass on both sides of the electrode tank. A gel mold is sandwiched between the two electrode tanks. The mold is composed of an upper frame-shaped gel frame, long and short glass plates, and a sample tank template (comb). Formed by. The electrophoresis tank is composed of an upper storage tank (the platinum electrode is on the top or facing the short glass plate), a lower storage tank (the platinum electrode is on the bottom or facing the long glass plate) and a corrugated condenser tube. The two electrode grooves and the gel mold are fixed by the liquid storage tank screws. The parts are assembled in the following order:
(1) Put the upper storage tank and fixing screw pin on the table top.
(2) Insert the upper and shorter glass plates into the concave grooves of the upper frame-shaped silicone rubber. Be careful not to touch the glass on the glued surface with your hands.
(3) Place the gel mold of the inserted glass plate on the upper storage tank, and the short glass plate should face the upper storage tank.
(4) Align the pin hole of the lower storage tank with the upper storage tank where the screw pin is installed, and tighten the screw cap diagonally with both hands.
(5) In the vertical electrophoresis tank, add the melted 1% agar (sugar) in the gap between the lower end of the long glass plate and the silicone mold frame. The purpose is to seal the gap, and avoid bubbles in the solidified agar (sugar).
2. With glue
â‘ Separation gel 20ml pH8.9 7.0% PAA solution, gel buffer (pH8.9) 2.5ml, separation gel stock solution 5.0ml, double distilled water 2.5ml, mix well, evacuate for 10min, and finally add AP 10ml .
â‘¡ Concentrated gel pH6.7 25% PAA: Concentrated gel buffer: concentrated gel stock solution: 40% sucrose solution: riboflavin solution in a ratio of 1: 2: 4: 1.
3. Preparation of gel plates
The discontinuous system uses separation and concentration gels with different pore sizes and pHs. Gel preparation should be performed in two steps.
â‘ Preparation of separation gel: According to the experimental requirements, select the final concentration of acrylamide. In this experiment, 20ml of pH8.9 7.0% PAA solution is needed. The method of adding gel is different from that of continuous system. The mixed gel solution is added to the narrow gap between the long and short glass plates with a slender-tip dropper, and the height of the added gel is about 1 cm from the lower edge of the comb teeth of the sample template. Use a 1cm syringe to gently add a layer of double-distilled water (about 3-4mm) along the edge of the short glass plate on the surface of the gel to isolate the air and make the glue surface flat. To prevent leakage, add distilled water slightly below the glue surface to the upper and lower storage tanks. After about 30-60min, the gel is fully polymerized, and you can see that the water and the solidified gel surface have different refractive index boundaries. Absorb excess water with a filter paper strip, but do not touch the rubber surface. If pre-electrophoresis is required, the distilled water in the upper and lower storage tanks is poured, and the separation gel buffer is replaced, and the electrophoresis is carried out at 10mA for 1h. After the electrophoresis is terminated, the separation gel buffer is discarded, and the concentrated gel buffer is used to wash the number of gel surfaces At this time, the concentrated gel can be prepared.
â‘¡ Preparation of concentrated gel: The concentrated gel is pH6.7 25% PAA. After mixing, add the gel solution to the narrow slits of the long and short glass plates (that is, above the separation gel) with a long and thin dropper. At the upper edge of 0.5cm, gently add the sample tank template. Add distilled water to the upper and lower storage tanks, but not beyond the upper edge of the short glass plate. At a distance of 10 cm from the electrode groove, use a fluorescent lamp or the sun to perform photopolymerization, but do not cause a large temperature. Under normal conditions, the gel is transparent from egg yolk to milky white when irradiated for 6-7min, indicating that polymerization has begun. Continue to illuminate for 30 min to complete the gel polymerization. After the photopolymerization and the completion, place it for 30-60min, gently remove the sample tank template, use a narrow strip of filter paper to suck up the excess liquid in the sample groove, and add Tris-glycine electrode buffer diluted 10 times pH8.3. The sample can be added when the liquid level is less than 0.5cm above the glass plate.
4. Add samples
The amount of PAGE sample used for analysis is only a few micrograms, and dozens of protein bands can be separated after 2-3ul of serum electrophoresis. To prevent the sample from spreading, an equal volume of 40% sucrose (containing a little bromophenol blue) should be added to the sample. Take 5ul of the above mixed solution with a micro syringe and carefully add the sample to the bottom of the concave sample tank of the gel through the buffer solution. After all the concave sample tanks are filled with samples, electrophoresis can be started.
5. Electrophoresis
Connect the positive pole of the DC stabilized electrophoresis instrument to the lower tank and the negative pole to the upper tank (do not connect them in the wrong direction). Turn on the cooling water, turn on the electrophoresis switch, and initially adjust the current to 10 mA. Adjust the current to 20-30mA when the sample enters the separation gel. When the blue dye migrates to 1cm from the lower edge of the rubber frame, adjust the current to zero, turn off the power and cooling water. Collect the electrode buffer solution of the upper and lower storage tanks respectively in the reagent bottle, and store at 4 ℃ for 1-2 times. Loosen the fixing screws, take out the silicone rubber frame, use a stainless steel shovel to gently pry off a glass plate, remove one corner of the rubber plate as a mark, and move the rubber plate to a large Petri dish for staining.
6. Fixing and dyeing
In this experiment, 0.05% Coomassie Brilliant Blue R250 (containing 20% ​​sulfosalicylic acid) dyeing solution was used. The dyeing and fixing were carried out at the same time, so that the dyeing solution did not pass through the rubber plate and was dyed for about 30 minutes.
7. Decolorization
Rinse with 7% acetic acid several times until the background blue fades. If you use 50 ℃ water bath or faded shaker, you can shorten the fade time. After the decolorizing liquid is decolorized by activated carbon, it can be used repeatedly.
(Four) matters needing attention
1. Use high-purity reagents to prepare the gel, otherwise it will affect the gel polymerization and electrophoresis effect. Acr and Bis are the key reagents for preparing gels. If acrylic acid or other impurities are contained, the gel polymerization time will be prolonged and the polymerization will be uneven or non-polymerized. They should be purified separately before use. Both Acr and Bis are nerve agents, which have a stimulating effect on the skin. Experiments have shown that the LD50 for mice is 170 mg / kg, and the operation should be carried out in ventilation. Purification of Acr: Weigh 70gArc in 1000ml chloroform preheated at 50 ℃, dissolve and filter while hot After cooling, put it in a -20 ℃ low temperature refrigerator, white crystals will precipitate out, filter with a pre-cooled Büchner funnel, collect the white crystals, rinse with pre-cooled chloroform several times, vacuum dry and seal in a brown bottle Save. The melting point of Acr is 84.5 + 0.3. The pH of the pure Acr aqueous solution should be such that its pH value does not change more than 0.4 pH units before it can be used. Purification of Bis: Weigh 12g Bis, dissolve it in 1000ml of acetone preheated at 40-50 ° C, and filter while hot. After cooling, place it in a -20 ° C low temperature refrigerator. After the crystals are precipitated, use a pre-cooled Büchner funnel to suction filter, collect the crystals, wash them with pre-cooled acetone several times, vacuum-dry and store in a brown bottle imam sealed, Bis melting point is 185 ℃. During storage, Acr and Bis stock solutions form acrylic acid and NH3 due to hydrolysis. Although the solution is placed in a brown reagent bottle, storage at 4 ° C can partially prevent hydrolysis, but it can only be stored for 1-2 months, measurable pH value (4.9-5.2) to check whether the reagent is invalid.
2. Because the surface of the silicone rubber strip and glass plate related to gel polymerization is not smooth and clean, it will cause the gel plate to peel off from the glass or silicon rubber strip during electrophoresis, resulting in bubbles or slip glue; the gel plate is easy to break when peeling off In order to prevent secondary phenomena, the equipment should be cleaned strictly. The groove of the silicone rubber strip, the sample tank template and the electrophoresis tank are carefully cleaned with a sponge sponge dipped in "clean clean". The glass plate is soaked in potassium dichromate wash solution 3-4h or 0.2mol / LKOH alcohol solution for more than 20min, washed with water, then dipped in a foam sponge to "wash clean" and repeatedly brushed, and finally rinsed with distilled water, directly Dry in the shade or rinse with ethanol.
3. When installing the electrophoresis tank and the silicone rubber frame with long and short glass, the position should be correct, and the fixing screw should be rotated tightly and evenly to avoid buffer leakage. The comb teeth of the sample slot template should be flat and smooth.
4. There should be no air bubbles when agar (sugar) is used for bottom sealing and gel filling, so as not to affect the current flow during electrophoresis.
5. After the gel is completely polymerized, it must be placed for 30min-1h to allow it to fully "age" before gently removing the sample tank template. Do not damage the flatness of the bottom of the loading groove to avoid distortion of the electrophoresis zone.
6. In order to prevent tailing after electrophoresis, the salt ion strength in the sample should be as low as possible. The sample tank with high salt content can be desalted by dialysis or gel filtration. The maximum sample volume should not exceed 100ug protein / 100ul.
7. In the discontinuous electrophoresis system, pre-electrophoresis can only be performed after the separation gel is polymerized, and the purulent gel can only be prepared after washing the gel surface. After the concentrated gel is prepared, pre-electrophoresis cannot be performed to make full use of the concentrated effect of the concentrated gel.
8. During electrophoresis, the positive and negative poles of the electrophoresis instrument and the electrophoresis tank must not be connected incorrectly to prevent the sample from swimming in the opposite direction. The appropriate current, voltage, too high or too low during electrophoresis can affect the electrophoresis effect.
9. After electrophoresis, the electrode buffer solution of the upper and lower storage tanks should be collected separately and stored in the refrigerator, which can be used 2-3 times. To ensure satisfactory electrophoresis results, it is best to use freshly diluted buffer.
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