Make useful antibody fragments from human or mouse IgM.

The Pierce IgM Fragmentation Kit easy generation of various immunoreactive fragments from 1 mg samples of mouse or human IgM. The kit contains all the necessary components for producing "IgG", "Fab" and "F(ab)2" type fragments from intact IgM using trypsin, pepsin or 2-mercaptoethylamine. For each protocol, approximately 1 mg of IgM is first prepared by buffer exchange with supplied gel filtration columns into an optimized digestion buffer. The IgM is then digested by one of the kit protocols to acheive the desired fragment type. C100 and C30 Concentrators in the kit allow fragments to be separated and concentrated for downstream applications.

Figure 1. Digestion strategies for preparing antibody fragments from intact IgM.

The trypsin and pepsin proteases are supplied immobilized on beaded agarose resin, eliminating the need to separate enzyme from IgM fragments. The IgM is digested as it passes through the pre-packed immobilized enzyme columns. Digestion can be easily regulated by digestion time or flow rate through the column. The enzyme remains bound to the support, ensuring that there is no auto-digested enzyme to contaminate the IgM fragments. The immobilized enzymes also offer increased stability against heat denaturation and autolysis and yield reproducible digests. The detailed instructions provide information about IgM structure, fragmentation, fragment nomenclature, digestion enzymes, reduction and alkylation.

Highlights

• Makes IgM antibodies more useful – fragmenting large IgM antibodies into smaller immunoreactive molecules provides for greater flexibility and ease of use for many immunochemical techniques, including labeling and immunohistochemical detection
• Enzyme-free digestion products – immobilized trypsin and pepsin enzymes (beaded agarose resins) provide for control of the digestion reaction and complete removal of resulting antibody fragments from the proteases
• For human or mouse IgM – the kit procedure is optimized for human and mouse IgM but the protocol can be adapted for use with other species and subclasses of IgM
• Flexible – kit provides all components needed to prepare the type of IgM fragment you choose

Related Products
Fab Preparation Kit - for IgG
F(ab')2 Preparation Kit - for IgG
Mouse IgG1 Fab and F(ab')2 Preparation Kit

References

1. Beale, D. and Van Dort, T. (1982). A comparison of the proteolytic fragmentation of immunoglobulin M from several different mammalian species. Comp. Biochem. Physiol. 71B(3): 475-482.
2. Chen, F.-M. and Epstein, A.L. (1988). Preparative separation of IgM monoclonal antibody from mouse ascites and purification of F(ab')2 fragments-the use of ABx and Superose columns, AB. Immunoconjugates, Radiopharmaceuticals 1(4): 333-341.
3. Poncet, P., et al. (1988). Immunochemical studies of polyspecific natural auto-antibodies: charge, lipid reactivity, F(ab')2 fragments activity and complement fixation. Mol. Immunol. 25(10): 981-989.
4. Kakimoto, K. and Onoue, K. (1974). Characterization of the Fv fragment isolated from a human immunoglobulin M. J. Immunol. 112(4): 1373-1382.
5. Lin, L.-C. and Putnam, F.W. (1978). Cold pepsin digestion: a novel method to produce the Fv fragment from human immunoglobulin M. PNAS 75(6): 2649-2653.
6. Herremans, T., et al. (2000). Clin. Diagn. Lab Immunol. 7(1): 40-44.