(a) principle The crude protein obtained by salting out (the IgG obtained by salting out) contains salts such as ammonium sulfate, which will affect the subsequent purification, so it should be removed before purification. This process is called "desalthing". . Desalination is commonly used in dialysis and gel filtration, both of which have advantages and disadvantages. The advantage of the former is that the final volume of the precipitate after dialysis is small, but the time required is long, and the salt is not easily removed; the gel filtration method can remove the salt and the time required is short, but the sample volume after gel filtration Larger. Therefore, you should choose to use according to the specific situation. In the previous experiment, the sample volume was small, and the sample volume of the gel did not increase too much after filtration, so the gel filtration method was selected. (2) Reagents and equipment (1) Sephadex G-25. (2) 0.0175 mol/L, pH 6.7 phosphate buffer. (3) Nessler reagent: 150 g of potassium iodide, 110 g of iodine, 150 g of mercury, and 100 ml of distilled water were placed in a 500 ml Erlenmeyer flask. Shake vigorously for 7 to 15 minutes. When the brown color of iodine begins to change, the temperature of the mixture rises. The bottle is immersed in cold water and continues to oscillate until the brown iodine is converted into a green potassium iodide solution. The supernatant was poured into a 2000 ml measuring cylinder, distilled water was added to 2000 ml, and mixed for use. (4) 20% (w/v) sulfosalicylic acid solution. (5) 1.5 cm × 20 cm column. (6) Black and white color disk. (three) operation (1) Take one column (1.5 cm × 20 cm), fix it vertically on the support, and close the lower end outlet. The water in the already swollen Sephadex G-25 was decanted, and 2 volumes of 0.0175 mol/L, pH 6.7 phosphate buffer was added and stirred into a suspension, which was then poured into the column and the lower end outlet of the column was opened. Continue to add the homogenized Sephadex G-25 so that the gel naturally settles to a height of about 17 cm and close the outlet. After the gel column was formed, 0.0175 mol/L, pH 6.7 phosphate buffer was added to the gel column with 3 column volumes of phosphate buffer to balance the gel. (2) After the gel is equilibrated, remove the solution of the gel cylinder with a straw dropper, add all IgG samples obtained by salting out to the surface of the gel column, open the lower mouth of the column, and control the flow rate to slowly immerse the IgG sample solution into the gel. Inside the glue. A layer of 0.0175 mol/L, pH 6.7 phosphate buffer was added to the gel column and eluted with this buffer to control the flow rate to about 0.5 ml/min. The eluate was collected in a test tube, 10 drops per tube. (3) Check if the protein has started to flow out while collecting the eluent. To this end, take 1 drop of solution from each collection tube and place it on a black colorimetric disk. Add 1 drop of 20% sulfosalicylic acid. If it appears as a white flocculent precipitate, it will prove that the existing protein appears until it can not be detected. At the time of precipitation, the collection of the eluate was stopped. (4) From each tube containing the protein to be examined, take 1 drop of the solution, place it in the white colorimetric well, add 1 drop of Nessler's reagent, and if it shows a brownish yellow precipitate, it contains ammonium sulfate. After the combined inspection, the tube collection liquid containing no ammonium sulfate is the IgG after "desalting". Note: During the test, the solution in the tube should be washed with a straw dropper, and then the next tube should be taken to avoid the mutual pollution. We are a professional Chinese manufacturer of Fat Loss Material ;we supply various products of Fat Loss Material , and can providing product images and basic parameters with each Lipase inhibitor and Boosting Metabolism Rate Material. We have the perfect after-sales service and technical support. Please do not hesitate to contact us, Look forward to your cooperation! Fat Burning Material,L-Carnitine 99%,Apple Cider Vinegar Powder,Raspberry Ketone,Capsaicin Powder Xi'an Quanao Biotech Co., Ltd. , https://www.quanaobiotech.com