Bulk desorption of IgG from protein A-Sepharose columns

Bulk desorption of IgG from protein A-Sepharose columns. SpA-Sepharose (1.5 g) is swollen in 10 mM phosphate-buffered saline (PBS), pH 8.0, at room temperature, to about 5 ml and poured into a small column (0.9 x 15 cm). This column may be stored with 0.1% NaNs at 4°C for long periods. The column is washed once with 100 mM sodium citrate buffer, pH 3.0, to elute any bound material, and then with PBS before use (3-4 volumes). Serum diluted with an equal volume of PBS is passed through the column at a rate of about 1 ml/3 min (capacity about 15 mg/ml). The absorbance of the effluent is monitored and buffer is passed until no more protein leaves the column. IgG is then eluted with 100 mM sodium 

Goat IgG2 IgGl Delacroix and Vaerman, 1979 Duhamel et al., 198 

Stepwise gradient desorption of IgG subclasses from protein 

A-Sepharose. Subclasses can often be isolated by this tech- 

For the isolation of mouse IgG subclasses (Ey et al., 1978), the SpA-Sepharose column (Section 7.1.9.1.1) is equilibrated with 100 mM phosphate buffer, pH 8.0. The serum (5 ml) is diluted with 2 ml of the same buffer and the pH is adjusted to 8.1 with 1 M Tris-HCl buffer, pH 9.0. This sample is then applied to the column and washed with 30 ml of the pH 8.0 buffer. The effluent contains more than 80% of the IgM and IgA. IgGl is then eluted with 30 ml 100 mM sodium citrate, pH 6.0. Most of the IgG2a (90%) is then eluted with a 100 mM citrate buffer, pH 5.5, and the rest at pH 4.5, but this risks some denaturation. Finally, IgG2b is eluted with 30 ml 100 mM citrate buffer, pH 3.5. The column is then regenerated and equilibrated as in Section 7.1.9.1.1. 

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