The purification of IgM is not as easy as that of IgG. IgM may be prepared in good yield and reasonable purity by precipitation with 40% saturated ammonium sulfate followed by gel filtration on Sepharose 6B or Sephacryl S-500. It is important to choose a gel filtration medium which includes IgM, so that it is chromatographed rather than simply dropped through the column (see Section 9.2.4). The choice of an appropriate gel filtration medium allows separation of IgM from high molecular weight lipoproteins and protein aggregates, which would emerge with IgM in Sephadex G-200. Even on an appropriate gel, there is likely to be some contamination of IgM with a2-macroglobulin (Mr 725 000). Contamination with a2-macroglobulin may be reduced by substitution of euglobulin precipitation (see Section 9.2.2) for ammonium sulfate in the first step. However, euglobulin precipitation causes some denaturation, as shown by the frequent difficulties in redissolving the precipitates. In addition, not all IgM proteins are euglobulins.
Jehanli and Hough (1981) have shown that monoclonal human IgM may be purified to homogeneity by filtration on Ultrogel AcA 34 followed by ion exchange chromatography on DEAE-Sepharose CL6B with salt gradient elution. The IgM was bound to the ion exchanger in 0.05 M phosphate/citrate buffer at pH 6.8, and eluted with a linear gradient to 0.1 M phosphate/citrate, pH 5.0. Recovery was approximately 70%. This procedure may become the method of choice, and would probably be applicable to mouse IgM without modification. It would be preferable to use Ultrogel AcA 22, which chro-matographs IgM, rather than AcA 34, which does not.
Inouye (1991) purified rat IgM from ascites by precipitation with 60% saturated ammonium sulfate, followed by gel filtration on TSKgel Toyopearl HW-55 (Tosoh, Tokyo, Japan) in 100 mM citrate buffer, pH 4.5.
There are numerous other published protocols for purification of IgM. Tatum (1993) published a large-scale method involving sequential precipitation with ammonium sulfate followed by resuspension and reprecipitation with polyethylene glycol. No chromatographic steps were needed. Other procedures for purification of IgM are given by Hayzer and Jaton (1985).
Knudsen et al. (1992) showed that thiophilic adsorption chromatography can be used to purify human and mouse IgM, although the purity was not assessed by polyacrylamide gel electrophoresis.
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