Protein Protocols Protein Protocols

1004 Page and Thorpe
7. Filter and wash the gel thoroughly. The gel may be stored at 4°C in 0.02% sodium azide.
8. Pack 4 mL of the gel in a polypropylene column (10 × 1 cm) and equilibrate with 25 mL of
binding buffer.
9. Perform chromatography at 4°C. Mix 1 mL of IgG containing sample with 2 mL of binding
buffer, and load onto the column.
10. After the sample has entered the gel, wash non-IgG from the column with 20 mL of binding
buffer; monitor the A 280 as an indicator of protein content in the wash until the absorbance
returns to background levels.
11. Elute the bound IgG with 0.1 M ammonium bicarbonate, and collect into 2-mL fractions.
Monitor the protein content by absorbance at 280 nm, and pool the IgG containing
fractions (i.e., those with protein absorbance peaks). Dialyze against an appropriate buffer
(e.g., PBS) with several changes, and analyze by gel electrophoresis under reducing
conditions (see Chapter 134).
4. Notes
1. The activated gel can be stored by washing thoroughly in acetone and kept as a suspension
in acetone at 4°C.
2. Immobilized ligands prepared by the divinylsulfone method are unstable above pH 8.0.
References
1. Porath, J., Maisano, F., and Belew, M. (1985) Thiophilic adsorption—a new method for
protein fractionation. FEBS Lett. 185, 306–310.