Isolation of murine and human immunoglobulin m and murine immunoglobulin D.

This unit describes two classical protocols for the purification of IgM-dialysis of ascites fluid, tissue culture medium, or bioreactor supernatants against distilled water to precipitate pure IgM, and ammonium sulfate precipitation. Both protocols can be followed by size-exclusion chromatography to obtain a highly purified product. Recently, an affinity method for purification of IgM has been developed using mannan-binding protein, and is described here.

Fragmentation of immunoglobulin M.

Fragmentation of IgM antibodies may be necessary because of the large molecular weight of the native molecule (900 kDa). IgMs fragments resemble IgG in size and structure, but they may have a decreased binding affinity. The Fc portion of IgM can have powerful biological effector functions such as complement activation.

Fragmentation of immunoglobulin G.

For some purposes, fragments of the IgG molecule are preferred. The F(c) portion is useful for studies of biological effect-binding to the F(c) receptor, mediating antibody-dependent cellular cytotoxicity, and complement fixation. The bivalent F(ab')(2) produced by digestion with pepsin and the monovalent Fab produced by digestion with papain are useful for studies based on the interaction between antibody binding site(s) with antigen.

Dialysis and concentration of protein solutions.

Conventional dialysis separates small molecules from large molecules by allowing diffusion of only the small molecules through selectively permeable membranes. Dialysis is usually used to change the salt (small-molecule) composition of a macromolecule-containing solution. The solution to be dialyzed is placed in a sealed dialysis membrane and immersed in a selected buffer; small solute molecules then equilibrate between the sample and the dialysate.