Purification Of IgM By Dialysis And Sizeexclusion Chromatography

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Many IgM antibodies will precipitate when dialyzed against distilled water. The IgM produced by this method is of sufficient purity to be used for fragmentation (unit 2.10A), fluorescein isothiocyanate (FITC) or biotin labeling (unit5.3), or radiolabeling (unit8.11).

The protocol involves centrifugation of ascites fluid or monoclonal antibody supernatant, followed by simple dialysis against water. IgM is separated from contaminating proteins (such as normal mouse proteins or proteins from fetal bovine serum) by use of size-exclusion (SE) chromatography. The purity of IgM is checked by SDS-PAGE and the pure IgM is stored in borate buffer.

Materials

Ascites fluid or monoclonal antibody (MAb) supernatant (unit 2.6) PBS (appendix 2)

Sorvall centrifuge and SS-34 rotor (or equivalent) Dialysis tubing (appendix 3h)

26 x 900-mm column with appropriate size-exclusion (SE) resin (appendix 3i)

Additional reagents and equipment for purification of immunoglobulin G (unit2.7), protein dialysis (appendix3h), size-exclusion chromatography (appendix 3i), and SDS-PAGE (unit 8.4)

1. Clarify ascites fluid by centrifugation and remove lipid as described for IgG purification in unit2.7 (step 1 of Basic Protocol 1). If using MAb supernatant, centrifuge 30 min at 15,000 x g (11,000 rpm in SS-34 rotor), room temperature or 4°C, to remove cell debris. Save supernatant.

2. Transfer clarified ascites fluid or MAb supernatant to dialysis tubing and dialyze (appendix3h) against >10 times the sample volume of distilled water for 24 hr at 4°C.

3. Centrifuge dialyzed material 1 hr at 15,000 to 20,000 x g (11,000 to 13,000 rpm in SS-34 rotor), room temperature or 4°C, and discard supernatant.

Contributed by Sarah M. Andrew, Julie A. Titus, Richard Coico, and Ashok Amin

Current Protocols in Immunology (1997) 2.9.1-2.9.8 Copyright © 1997 by John Wiley & Sons, Inc.

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