Immunoaffinity Purification of Antibodies

Immunoaffinity purification of antibodies on an antigen column (SEK 12/12/95)

1. Wash antigen column as follows:

5 ml 100 mM Tris, pH 8.0

5 ml 100 mM glycine, pH 2.5 (prepared fresh)

5 ml 10 mM Tris, pH 8.0

5 ml 100 mM triethylamine (TEA), pH 11.5 (prepared fresh)

2x 5 ml 100 mM Tris

2. Pass 5 ml of IgG eluate through affinity column, 10 times at a slow flow rate (about 10 min/pass) for 1–1.5 h.

3. Collect flow thru; save.

4. Wash the column with:

2x 5 ml of 10 mM Tris, pH 8.0

2x 5 ml of 10 mM Tris and 500 mM NaCl, pH 8.0

5. Elute with 4.5 ml 100 mM glycine, pH 2.5. Collect the eluate in a tube containing 0.5 ml 1 M Tris, pH 8.0 and 1 ml of 50 mg/ml BSA (=10 mg/ml BSA final).

6. Wash column with 2x 5 ml 100 mM Tris, pH 8.0 until pH rises to 8.0.

7. Elute with 4.5 ml 100 mM TEA, pH 11.5. Collect the eluate in a tube with 0.5 ml 1 M Tris, pH 8.0 and 1 ml of 50 mg/ml BSA.

8. Wash column with 2x 5 ml of 100 mM Tris.

9. Wash column with 5 ml 100 mM Tris and 0.02% NaN3 until only 1 ml remains at top.

10. Combine both acid and base washes.

11. Dialyze combined antibody fractions and saved flow thru fractions overnight against 3x 2 L PBS at 4°C (all 3 i.e., R-4586 IgG fraction, R-4586 flow thru, R-4586 bound).

12. Add sodium azide to a final concentration of 0.02% and store these antibodies at 4°C.