The Thermo Scientific Pierce IgM Purification Kit is an easy-to-use affinity matrix and buffer system to purify IgM from mouse ascites fluid; the method uses immobilized mannan binding protein, which binds to IgM in the kit-supplied buffers.
Pierce MBP Agarose is purified mannan binding protein (also called mannose-binding lectin, MBL) that has been immobilized on 4% beaded agarose. When used with an optimized buffer system based on temperature- and calcium-dependent binding, this resin enables specific affinity purification of mouse IgM from ascites fluid and other concentrated samples. The resin and essential buffers are available individually or bundled together in a complete IgM Purification Kit containing a pre-packed 5mL column of the MBP Agarose.
- Immobilized MBP – affinity system based on mannan binding protein (MBP) allows specific purification of mouse IgM from ascites fluid or partial isolation of human IgM from serum
- Complete kit – IgM Purification Kit includes a pre-packed MBP column and ready-to-use buffers for convenient purification of at least 1.5mg IgM from 0.5mL ascites samples per column-use
- Mild conditions – nondenaturing, near-neutral buffer conditions ensure recovery of functional antibodies
- Reusable – stable ligand and resins can be regenerated and reused at least 10 times without significant decline in binding capacity
Mannan binding protein (MBP), also known as mannose-binding lectin (MBL) is a mammalian lectin that is produced in the liver and occurs in serum. MBP consists of 18 identical subunits, each with a molecular mass of approximately 31kDa. The protein is capable of initiating carbohydrate-mediated complement activation.
The IgM Purification Kit protocol based on immobilized MBP is most effective for purifying mouse IgM from ascites fluid, although it also enables partial purification of human IgM from serum. Binding and elution steps are temperature- and calcium-dependent. Binding and washing steps are performed at 4°C in Tris buffer that contains 20mM calcium chloride. Elution is achieved at room temperature in Tris buffer that contains EDTA but no calcium chloride. The simple protocol is easy to use and yields 90% pure mouse IgM from ascites. Immobilized MBP can be regenerated at least 10 times with no apparent loss of binding capacity. Human IgM also will bind to the support but with slightly lower capacity and some copurification of IgG. The purification system has not been tested or validated for use with other species and sources of samples.
- 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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