Purification Of IgM By Ammonium Sulfate Precipitation

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This protocol can be used for most IgM antibodies. It is more time-consuming and tedious than dialysis against water, but should be used for IgM antibodies that will not precipitate in water. Ammonium sulfate precipitation of IgM is similar to that described for IgG in unit2.7 (Basic Protocol 1). This is followed by dialysis and size-exclusion chromatogra-phy as described above for IgM.

Additional Materials (also see Basic Protocol 1) Saturated ammonium sulfate (SAS; unit 2.7) Ammonium sulfate crystals (CAS)

Additional reagents and equipment for purification of IgG (unit2.7) and size-exclusion chromatography (appendix 3i)

Follow step 1a or 1b in unit2.7 (Basic Protocol 1) to prepare ascites fluid or MAb supernatant, respectively.

For precipitation of protein from ascites: Add SAS to 45% (v/v; unit 2.7, Basic Protocol 1, step 2).

For precipitation of protein from MAb supernatant: Add the following number of grams of crystalline ammonium sulfate (CAS) to the supernatant:

For example, starting with 100 ml of supernatant, 34.2 g CAS should be added.

Proceed with precipitation and concentration of the protein solution (unit2.7, Basic Protocol 1, steps 3 to 6).

Prepare and run an SE column (appendix 31), and then analyze and store the purified IgM, as in the IgM dialysis method (see Basic Protocol 1, steps 4 to 7).

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