1. Dialyze about 1mg of purified His-tagged antigen in coupling buffer. This step can be done as the last step in His-tag purification procedure.
2. Weigh out 0.25g of CNBr-activated sepharose powder and swell in 5ml of 1mM HCl in 15ml Falcon tube.
3. After the powder swells into beads, add 1mM HCl to 10ml and nutate at RT for ~5min. Spin at 1000rpm for 1min. and remove the supernatant. Repeat washes at least 5 times with 10ml 1mM HCl.
4. After final wash remove all HCl and add 1mg of His-antigen dissolved in about 2.5ml of coupling buffer.
5. Nutate overnight at 4 deg.C or at RT for 1 hr.
6. Spin at 1000rpm for 1min. and remove the protein solution from activated beads.
7. Wash beads with 5ml of coupling buffer to wash away excess ligand.
8. Add 5ml of 0.1M Tris-HCl pH 8.0 to the beads and allow to stand at RT for 2 hours to block any remaining active groups.
9. Mix beads and load the slurry onto a column and allow Tris to drain.
10. Wash column three times alternatively with 5ml of low pH and high pH wash solutions.
11. Wash column with 5ml of 10mM Tris pH 7.5.
12. Dilute 2ml of anti-serum with 18ml of 10mM Tris pH7.5 and add pass it over the column (collect the flow through).
13. Wash column twice with 15ml of 10mM Tris pH7.5 and once with 10mM Tris pH 7.5 and 0.5M NaCl.
14. Elute bound antibodies with 250ul of 100mM Glycine pH 2.5 into 25ul of 1M Tris pH8.0 in eppendorf. Repeat elutions until protein is detected in eluates as estimated by bench-Bradford.
15. Wash column with 10mM Tris pH 8.0 until the pH of flow through is 8.0 (about 10ml).
16. Elute with 250ul of 100mM triethylamine into 25ul of 1M Tris pH 8.0. Repeat elutions until protein is detected in eluates as estimated by bench-Bradford.
17. Pool all eluted fractions that contain significant amount of protein and dialyze against PBS overnight at 4 deg. C

Coupling Buffer
0.1M NaHCO3 pH 8.3
0.5M NaCl

Low pH Wash Solution
100mM Glycine-HCl pH 2.5
0.5M NaCl