Chromatography of globulin
When adding neutral elegance to necessary concentration in the solution, the protein will sink and precipitate, and the salt precipitated protein will be in its body, which can be dissolved after dialysis or dilution. Salting is a reversible process. The tember-globulin was precipitated, so the tember-globulin could be distinguished and then purified by gel filtration. In serum proteins, albumin plays an important role. In addition, the chromatographic process of globulin, when the serum is involved in ammonium sulfate to 33% sufficient, this infection is called salting. The charge on a protein molecule is neutralized. 1. Salting of serum protein
(1) 1.0ml of serum was added to the centrifuge tube, followed by 1.0ml of PBS, mixed, and then interference drop by drop, pH7.2 saturated ammonium sulfate was added and shaken, and centrifuged at 3000rpm for 10min after 30min, and the supernatant was poured (this solution mainly contains albumin).
(2) the precipitation in the centrifuge tube was stirred and dissolved with 1.0ml PBS solution, and then mixed with 0.5ml saturated ammonium sulfate solution one drop at a time. The product was centrifuged at 30min and 3000rpm for 10min, and the supernatant was poured (mainly containing Chinese, Chinese ginseng). To win a simpler case of confection - globulin, go through this again and again.
2. Use chromatography equipment for desalting
(1) pretreatment of gel products: take Sephadex G50 1g, insert it into 100ml beaker, add 50ml distilled water, boil it for 1h in the open water bath, pour the supernatant after cooling, and prepare for attendance at PBS 10ml.
(2) loading column: the suspension of Sephadex G50 was mixed and presented at the top of the column for stagnation. When the gel precipitated 1-2cm high in the column, the outlet was opened and the gel was involved until it was controlled 2cm from the top. The column bed was washed continuously with PBS, and its flow disease was monopolized as 1ml/min. Beware of bubbles or layering during mastery. If the bed surface is unyielding, use glass rod to stir the upper layer gently so that the gel can be settled from the top of the head (throughout the process, do not make the liquid level below the bed surface, lest the gas enter, making the column bed dry crack).
(3) add sample: the liquid above the bed shall be released, and the liquid surface shall be ready to be weighed with the gel surface, and the lower port shall be closed. Use a dropper to absorb styrene-globulin solution, awkwardly coming in along the inner wall (try not to disturb the gel surface). Open the outlet to allow the sample to enter the column, then continue to be careful to add PBS.
1. Chromatography column 2. Latex tube 3. Screw clamp 4. Rubber plug
1. Serum samples. Shanghai chuangsai technology supply procedure fetal bovine serum, commodity number: b11-s9030-200ml, cost 360 yuan.
2. PH7.2 drum and ammonium sulfate: adjust the saturated ammonium sulfate solution to pH7.2 with ammonia water.
(4) gather: prepare two answering boards, add 1 drop of all-inclusive solution to each hole, and add one drop of nats solution to each hole of one plate, NH4+ is yellow to orange. Design 12 small test tube network effluent, starting from the addition of sample, each tube for 1ml. After the liquid was measured at 280nm wavelength, elution volume was taken as abscissa and light density as ordinate to make the elution map.
(5) detection: add a drop of biuret reagent into each hole of another reverberation board, visit the biuret feedback and record the color depth, and use (- or +) implied. PDF
4. Nasreagent. Shanghai chuangsai technology co., LTD. Supplies naproxen reagent A liquid, commodity number: c49-14205-100ml, the cost is 266 yuan.
5. Biuret reagent: cuso4.5h2o 0.39g, sodium potassium tartrate 1.2g identified solution in 50ml water, 2.5n NaO60ml, KI 0.2g mixed with the above solution, and added water to 200ml.