Protocol for the Isolation of IgG by Ammonium Sulfate Precipitation
GUIDELINE
Antisera prepared by immunizing an animal with an antigen is a very complex mixture that includes all of the components of the serum. To concentrate and increase the potency of the antibody, it is usually necessary to isolate and purify the immunoglobulins. γ globulin (IgG) is the main component of serum immunoglobulins, accounting for about 75% of all immunoglobulins, so the isolation and purification of the antibody mainly involves the isolation and purification of IgG.
METHODS
- Take 20 ml of serum, add 20 ml of saline, then add 10 ml of (NH4)2SO4 saturated solution drop by drop to make 20% (NH4)2SO4 solution. Stir while adding, mix thoroughly, and leave for 30 min.
- Centrifuge at 3000 r/min for 20 min and discard the precipitate to remove fibrin.
- Add another 30 ml of ((NH4)2SO4 saturated solution to the supernatant to make a 50% (NH4)2SO4 solution, mix thoroughly and leave for 30 min. Centrifuge at 3000 r/min for 20 min, discard the supernatant.
- Add 20 ml of saline to the precipitate to dissolve it, and then add 10 ml of (NH4)2SO4 saturated solution to make 33% (NH4)2SO4 solution. After mixing thoroughly, let stand for 30 minutes.
- Centrifuge at 3000 r/min for 20 min and discard the supernatant to remove albumin. Repeat the previous step 2 times.
- Dissolve the precipitate with 10 ml of saline and put it into a dialysis bag.
- Dialyze to remove salt, dialyze in normal water overnight, then dialyze in saline at 4°C for 24 h, with several fluid changes in between.
- Centrifuge to remove precipitation (remove hetero-protein), the supernatant is the crude IgG.
- The supernatant is the crude extracted IgG, which was passed through a DEAE-cellulose chromatography column. Elute with 0.01 mol/L pH 7.4 PBS (0.03 mol/L NaCl) and collect the eluate.
- Proteins and their quantitative identification.
NOTES
Ammonium sulfate is preferable to the best quality because the inferior product contains a small amount of heavy metals that affect protein sulfhydryl groups. If the heavy metals must be removed from the inferior product, pass H2S into the solution, let it stand overnight, then filter it and heat to evaporate the H2S.
