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Star Republic: Guide for Biologists

Purification --- Protein A column

Edited by Chang Zhu
1. 1 ml of agarose>protein A-agarose binds 10-20 mg of antibody. Serum antibody concentration ~10 mg/ml, tissue culture supernatants 20-50 mg/ml, and ascites 1-10 mg/ml.

2. Equilibrate column with 20 bed volumes of 100 mM Tris-HCl (pH8.0).

3. Centrifuge the antibody solution to clear the sample.

4. Adjust pH to 7.5-8.0 by adding 100 mM Tris-HCl (pH 8.0).

5. Apply the sample to the protein A column. Let it flow through.

6. Wash the column with 10 bed volumes of 10 mM Tris-HCl (pH 8.0). Check no protein is detected in the flow through.

7. Elute the bound antibody with 100 mM glycine (pH 3.0). Collect 200-500 ml fractions. Elute into tubes that contain 20% of the fraction size of 1 M Tris-HCl (pH8.0).

8. Identify the antibody faction by estimating protein concentration using UV spectrometry or other methods.

9. Change buffer to PBS by dialysis or buffer exchange column.

10. To reuse the column, wash the column with 10 bed volumes of 100 mM glycine (pH 3.0), then 10 bed volumes of 100 mM Tris-HCl (pH8.0). Store in 100 mM Tris-HCl (pH8.0)/0.1% sodium azide.