ã€Fundamental】
When a neutral salt is added to a protein solution to a certain concentration, the protein will precipitate and precipitate. This effect is called salting out. Its mechanism is related to the following factors: the surface of the protein molecule's surface hydration layer is destroyed; the charge of the protein molecule is neutralized . The protein precipitated by salting out is of the same nature and can be dissolved after dialysis or dilution. Salting is a reversible process. In albumin, albumin accounted for the vast majority of the rest of the globulin, when the ammonium sulfate was added to the serum to 33% saturation, γ-globulin was precipitated, so that the γ-globulin can be isolated, and then used The gel filtration method removes salt to obtain relatively pure γ-globulin.
ã€equipment】
1. Column
2. Latex tube
3. Screw clamp
Rubber stopper
ã€Reagents】
1. Serum samples. Shanghai Genesis Technology provides standard fetal bovine serum, Standard Fetal Bovine Serum, product number: B11-S9030-200ml, price 360 ​​yuan.
2. pH 7.2 saturated ammonium sulfate: The saturated ammonium sulfate solution was adjusted to pH 7.2 with aqueous ammonia.
3. Phosphate buffer-physiological saline (PBS): 0.9% NaCl solution in 0.01 mol/L phosphate buffer (pH 7.2). Shanghai Chuangsai Technology provides 7601-54-9, sodium phosphate monobasic for HPLC, ≥ 99.0%, product number: C13-17844-50g, price 586.62 yuan.
4. Nessler's reagent. Shanghai Genesis Technology provides Ness reagent solution A, product number: C49-14205-100ml, price 266 yuan.
5. Biuret reagent: CuSO4.5H2O 0.39g, potassium sodium tartrate 1.2g were dissolved in 50ml water, 2.5N NaO60ml, KI 0.2g and the above solution were mixed, add water to 200ml.
ã€Steps】
1. Salting out of serum protein
(1) Add 1.0 ml of serum to a centrifuge tube, add 1.0 ml of PBS solution, and mix well. Add dropwise pH 7.2 saturated ammonium sulfate 1.0 ml, add it while shaking, stand for 30 min, centrifuge at 3000 rpm for 10 min, and pour it on Clear (this solution contains mainly albumin).
(2) Dissolve the precipitate in the centrifuge tube with 1.0 ml PBS solution, then add 0.5ml of saturated ammonium sulfate solution dropwise, stand for 30min, centrifuge at 3000rpm for 10min, and pour off the supernatant (mainly containing α, β globulin The precipitate is the preliminarily purified gamma globulin. To obtain a more pure gamma globulin, repeat this process 1-2 times.
Desalination
(1) Pretreatment of gel preparation: Take Sephadex G50 1g into a 100ml beaker, add distilled water 50ml, boil in boiling water bath for 1h, cool and dump the supernatant, add PBS 10ml again.
(2) Packing column: Close the outlet and mix the Sephadex G50 suspension from the top of the column. When the gel is 1-2cm high in the column, open the outlet and add the gel continuously until it is about 2cm from the top. . Continue washing the entire bed with PBS, controlling the flow rate to 1 ml/min. Avoid bubbles or delamination during operation. If the bed surface is uneven, a glass rod can be used to gently agitate the surface layer to allow the gel to re-set naturally (do not allow the liquid surface to fall below the bed surface during the entire process so as to prevent gas from entering and the column bed from cracking).
(3) Add sample: discharge the liquid above the bed surface, and when the liquid surface coincides with the gel surface, close the lower mouth. Use a pipette to draw the γ-globulin fluid slowly along the inner wall (try not to disturb the gel surface). Open the outlet and allow the sample to enter the column. Carefully add PBS solution.
(4) Collection: Prepare 12 vials to collect the effluent. From the start of addition, collect 1 ml per tube. After the collection solution was measured at a wavelength of 280 nm, the elution volume was plotted on the abscissa, and the optical density was plotted on the ordinate.
(5) Detection: Prepare two reaction plates, add 1 drop each of the collection solution to each well, and add one drop of Nafion solution to each well of one plate. NH4+ is yellow to orange. A drop of each of the biuret reagents was added to each well of the other reaction plate to observe the biuret reaction and record the color shade, indicated by (- or +). PDF
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