The majority of the immunoglobulins in serum will precipitate at ammonium sulphate concentrations equivalent to 50% saturation. This concentration of ammonium sulphate can be achieved either by adding the required weight of ammonium sulphate or by adding an equal volume of a saturated solution of the salt. The former procedure can be quicker in that the prior preparation of a saturated solution is not required. With the latter method, it is easier to avoid the localized occurrence of high concentrations (>50% saturation) of ammonium sulfate, which may result in the precipitation of additional protein species (see Note 9).
1. If necessary, clarify the serum by centrifugation at 4000g for 30 min at 4°C.
2. While gently stirring the serum, slowly add an equal volume of saturated ammonium sulfate. Alternatively, add 3.1 g of solid ammonium sulphate per 10 mL of serum. Again, the ammonium sulphate should be added slowly with gentle stirring. Continue stirring the solution at 4°C for at least 1h (see Note 10).
3. Centrifuge at 3000g for 30 min at 4°C and retain the precipitate.
4. The precipitate can be kept at 4°C if not required immediately or at -20°C for longer-term storage. Alternatively, the precipitate should be resuspended in a 50% volume of a suitable buffer, then dialyzed overnight at 4°C (see Note 11).
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