| IgM binds poorly to Protein A and Protein G making one-step IgM purification difficult. Standard methods for IgM purification generally are multi-step, tedious processes, or they are not effective for removing all of the major impurities present in IgM samples.
Pierce has developed an easy-to-use affinity matrix and buffer system to purify IgM. This matrix, an immobilized mannan binding protein (MBP) support, is most effective for purifying mouse IgM from ascites. Purified IgM can be obtained from a single pass over the affinity column. Human IgM will bind to the support and yield a product at least 88% pure by HPLC upon elution. The purification of IgMs from other species and mouse serum has not been optimized. Studies in Pierce labs show that MBP does not bind F(ab´)2 and Fab fragments.
Mannan Binding Protein is immobilized on two supports: agarose and UltraLink Biosupport. The Immobilized MBP is also available in a kit format. The kit contains sufficient buffers to perform 10 purifications using a 5 ml column of Immobilized MBP. Pierce’s IgM Purification Kit is easy-to-use and yields 90% pure mouse IgM (from ascites) with a very simple protocol. Immobilized MBP can be regenerated at least 10 times with no apparent loss of binding capacity. The figure below demonstrates the high purity of IgM purified from mouse ascites using Immobilized MBP.
IgM purified from mouse ascites using Immobilized MBP is at least 88% pure.
The bound material from mouse ascites was eluted from the 5 ml MBP column as described in the Standard Protocol. The highest 280 nm absorbing fraction from the elution was chromatographed using the conditions in the instruction booklet.
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References
- Nethery, A., Raison, R. and Easterbrook-Smith, S. (1990). Single-step purification of immunoglobulin M on C1q-Sepharose. J. Immunol. Meth. 126, 57-60.
- Ohta, M., Okada, M., Yamashina, I. and Kawasaki, T. (1990). The mechanism of carbohydrate-mediated complement activation by the serum mannan-binding protein. J. Biol. Chem. 265, 1980-1984.
- Nevens, J.R., Mallia, A.K., Wendt, M.W. and Smith, P.K. (1992). Affinity chroma-tographic purification of immunoglobulin M antibodies utilizing immobilized mannan binding protein. J. Chrom. 597, 247-256.
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