experimental method
- Ammonium sulfate salting out
| Principle of experimental method | To separate and purify the protein, the protein of interest is first extracted from the raw material, and then a large amount of impurities are removed by a crude separation method, and finally finely separated to obtain a protein of interest. In this experiment, the novel recombinant human TNF was used as an example to elucidate the extraction and preliminary separation of recombinant protein TNF. The extraction of TNF is a process of lysing the fermenting cells, releasing the extracted TNF in a certain condition and solution, and collecting the TNF component extracted by the mixture by centrifugation. The extract solution containing TNF contains a large amount of heteroprotein, and the solubility of the protein in the aqueous solution mainly depends on the number of water molecules on the surface of the protein molecule, that is, the degree to which the hydrophilic group on the surface of the protein forms a hydrated film with water molecules and the charge. In the case where a neutral salt such as ammonium sulfate is added to a protein solution, the affinity of the neutral salt to the water molecule is greater than that of the protein, so that the hydrated membrane around the protein molecule is weakened or disappeared, the solubility of the protein is lowered, and the neutral salt is added. The ionic strength of the protein solution changes, the surface charge of the protein is largely neutralized, and the solubility of the protein is further reduced, so that the protein molecules aggregate and precipitate together. Different proteins form precipitates in different concentrations of salt due to their different properties such as chargeability and hydrophilicity. According to this principle, the mixed protein can be roughly separated. In this experiment, most of the heteroprotein was removed by ammonium sulfate salting out to obtain preliminary purification of TNF. |
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| Experimental Materials | Novel gene recombinant human TNF |
| Reagents, kits | Sucrose TRIS Hydrochloride Ethylenediaminetetraacetic Acid Disodium Sodium Hydroxide STE Solution Dnase I Sodium Deoxycholate Solution |
| Instruments, consumables | Dialysis bag stir bar sterilization pot electronic balance beaker weighing paper spoon measuring tube beaker ice machine |
| Experimental procedure | First, reagent preparation 1. STE (25% sucrose 10 mmol/L Tris-HCl pH 8.5 1 mmol/L EDTA) Sucrose 250 g 1 mol/L Tris.HCL 10 ml 0.5 mol/L EDTA 2 ml, add water to 1 000 ml 115 ° C, 20 min autoclave 2. 1mol/L MgCl 2 Take MgCl 2 203.30 g, add water to 1 000 ml 3. Tris-HCL pH 8.5 Take Tris 121.14 g Adjust the pH to 8.5 with HCL and add water to 1 000 ml (12 mol/L HCL approx. 30 ml) 4. 0.5 mol/L EDTA pH 8.5 Disodium edetate 186.12 g NaOH 20 g, add water to 1 000 ml 121 ° C, 20 min autoclave Second, the operation steps 1. Weigh the bacteria, add 5~10 ml/g of bacteria to the STE solution, and stir until the cells are completely suspended. 2. Add 0.5~1 mg/g of bacteria to the lysozyme solution freshly prepared with STE solution, stir vigorously with a glass rod, and place in a 4 °C environment for 20 min. At this time, the bacterial liquid is viscous. 3. Add 10 mg/g of bacteria to the sodium deoxycholate (DOC) solution freshly prepared with STE solution and stir vigorously. 4. Add 5~10 ml of 1 mol/L MgCl 2 solution, stir well, add about 40 μl Dnase I (25 mg/ml) and stir until the bacterial solution becomes thin. 5. Centrifuge at 15 000 rpm for 30 min at 4 °C. 6. Centrifuge the supernatant, accurately measure its volume, determine the protein concentration, pour into a beaker placed in an ice bath, and slowly add solid ammonium sulfate to achieve 50% saturation while stirring. After the solid ammonium sulfate was dissolved safely, it was left to stand in an environment of 0 ° C for 30 min. 7. Centrifuge, 15 000 rpm, 4 ° C, 30 min. The supernatant was taken, the volume was measured, and the protein concentration was measured. 8. Centrifuge the supernatant into a dialysis bag and dilute at pH 8.5 20 mmol/L Tris-Hcl and 1 mmol/L EDTA solution at 4 °C. Add ammonium sulfate, whether it is solid ammonium sulfate or saturated ammonium sulfate solution. When adding, stir while adding. Pay special attention to 1. Add ammonium sulfate slowly, because it will cause coprecipitation of protein too quickly. The ammonium sulfate is ground into a powder, and the saturated ammonium sulfate solution is added dropwise one by one; 2 the stirring is slow, the stirring is vigorous, and the protein solution is filled. |
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