The octanoic acid extraction method can be used to isolate and extract Ig from human, rabbit and mouse serum and McAb from hybridoma-induced ascites and culture supernatants.
Operation method
Octanoic acid extraction
Materials and Instruments
Animal serum samples Move I. Materials and reagents For more product details, please visit Aladdin Scientific website.
Octanoic acid Acetic acid buffer PBS
Dialysis bags High-speed cryogenic centrifuge
1. Octanoic acid
2. 60 mmol/L, pH 4.0 acetate buffer, PBS
3. Dialysis bags
4. High-speed cryogenic centrifuge
II. Operational steps
1. The sample to be extracted with 60 mmol / L, pH 4.0 acetate buffer diluted 3 to 4 times, adjust the pH to 4.5, the specimen containing high lipid can be used silica powder or glass fiber adsorption method to remove lipids.
2. Slowly add n-octanoic acid while stirring at room temperature, 25 μl per ml of sample, or 30 μl per ml of sample if the sample volume is less than 5 ml, and stir for 30 min.
3. 10 000 × g centrifugation for 30 min, take the supernatant through the qualitative filter paper filtration, loaded with dialysis bag, with 20 times the volume of PBS, 4 ℃ dialysis over the liquid, the middle of the liquid change 3 ~ 4 times.
4. Then add 0.27 g of ammonium sulfate per mL of mixture and stir for 30 min.
5. Centrifuge at 5,000 g for 15 min, collect the precipitate, dissolve it in a small amount of PBS, and then centrifuge at 15,000 × g for 20 min. The supernatant is the extracted Ig, most of which is IgG, accounting for about 90%, and a small amount of IgA and IgM, with a recovery rate of >80%. The whole operation can be completed within 24 h.