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We use saturated ammonium sulphate solution kept at 4C and filtered directly before use.
A volume of the saturated ammonium sulphate representing 82% of the serum volume is reserved and kept at 4C. After the serum has been spun for 30min at 4500rpm at 4C, the supernatant is ready for the IgG enrichment.
A 1/5th part of the reserved cold ammonium sulphate is added to the serum every two minutes at 0,2,4,6 and 8 minutes respectively. Then incubation continues overnight at 4C.
The mixture is spun for 30 min at 4500rpm at 4C and the supernatant is decanted. The pellet is dissolved in half the original serum volume of deionized water at room temp and cooled down for 30min at 4C.
In the meanwhile a volume of the saturated ammonium sulphate representing 41% of the original serum volume is reserved and kept at 4C.
A 1/5th part of the reserved cold ammonium sulphate is added to the protein mix every two minutes at 0,2,4,6 and 8 minutes respectively. Then incubation continues for 30 min before the mix is spun as above.
Again, the supernatant is removed and the pellet is now dissolved in 1/3 of the original serum volume of PBS/0.02%NaN3.
The IgG-enriched fractions are stored in 50ml tubes at 4C until ready for affinity purification.