Science Gateway > Protocols > Cell Biology Protocols - Table of Contents

Pubplus.org - a PubMed search engine
Star Republic: Guide for Biologists

Purification --- affinity HiTrap

Edited by Chang Zhu
This protocol uses the 1 ml HiTrap NHS-activated column from Amersham Pharmacia Biotech.

1. Prepare the column by washing with 10 ml of 50%, 25%, 10% Isopropanol, and then 10 ml 1 mM ice-cold hydrochloric acid (HCl).

2. Resuspend or dilute peptide or protein in the coupling buffer (0.2 M NaHCO3, 0.5 M NaCl, pH 8.3) to ~ 5 mg/ml. Apply 0.5 ml to the HiTrap column and hold at the room temperature for 1 hour.

3. Wash with 5 ml of the coupling buffer (0.2 M NaHCO3, 0.5 M NaCl, pH 8.3).

4. Wash alternately with 5 ml of 10 mM Tris-HCl pH8.3/0.5 M NaCl and 100 mM Sodium acetate pH 4.0/0.5 M NaCl three times.

5. Equilibrate the column with 10 mM Tris-HCl pH7.5, 0.1 mM EDTA, 50 mM NaCl, 0.01% sodium azide.

6. Apply the antibody sample and hold at room temperature for 10 minutes.

7. Wash with 50 ml of 10 mM Tris-HCl pH7.5.

8. Wash with 50 ml of 10 mM Tris-HCl pH7.5/0.5 M NaCl.

6. Elute with 5 ml 100 mM glycine, pH 2.5-2.8. Collecting 0.2-0.5 ml fractions. Immediately neutralize with 1/10 faction volume of 1 M Tris-HCl pH 8.0 in the collection tubes.

7. Read A280 and pool antibody containing fractions.

8. Dialyze against PBS/0.02% sodium azide.

9. The affinity column can be regenerated with extensive wash of 0.2 M glycine (pH 2.8), followed by extensive wash of a pH 7.0-8 buffer.