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The use of size-exclusion chromatography (SEC; also known as gel filtration chromatography) for purification of IgG has been described in protocol 131. SEC with dextran, agarose, and polyacrylamide soft gels was one of the first techniques developed for the purification of proteins. Soft gels, however, cannot tolerate high pressure and require long separation times. They also have limited stability to extremes of pH and to salt. These and other limitations of "conventional" size-exclusion chromatography are responsible for the problems with resolution often observed when analyzing/ purifying IgG using such methods. However, many of these problems can be solved by using high performance liquid chromatography (HPLC) systems with appropriate sizeexclusion columns. [ABSTRACT FROM AUTHOR]

Conventional ion-exchange chromatography separates molecules by adsorbing proteins onto the ion-exchange resins that are then selectively eluted by slowly increasing the ionic strength (this disrupts ionic interactions between the protein and column matrix competitively) or by altering the pH (the reactive groups on the proteins lose their charge). Anion-exchange groups (such as diethylaminoethyl; [DEAE]) covalently linked to a support matrix (such as Sepharose) can be used to purify IgG in which the pH of the mobile-phase buffer is raised above the pI or IgG, thus allowing most of the antibodies to bind to the DEAE matrix. Compare this method with that described in Chapter 139 in which the IgG passes through the column. The procedure can be carried out using a laboratory-prepared column that is washed and eluted under gravity (1); however, high-performance liquid chromatography (HPLC) provides improved reproducibility (because the sophisticated pumps and accurate timers), speed (because of the small high-capacity columns), and increased resolution (because of the fine resins and control systems). [ABSTRACT FROM AUTHOR]