- 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.
- Weigh out 0.25g of CNBr-activated sepharose powder and swell in 5ml of 1mM HCl in 15ml Falcon tube.
- 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.
- After final wash remove all HCl and add 1mg of His-antigen dissolved in about 2.5ml of coupling buffer.
- Nutate overnight at 4 deg.C or at RT for 1 hr.
- Spin at 1000rpm for 1min. and remove the protein solution from activated beads.
- Wash beads with 5ml of coupling buffer to wash away excess ligand.
- 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.
- Mix beads and load the slurry onto a column and allow Tris to drain.
- Wash column three times alternatively with 5ml of low pH and high pH wash solutions.
- Wash column with 5ml of 10mM Tris pH 7.5.
- Dilute 2ml of anti-serum with 18ml of 10mM Tris pH7.5 and add pass it over the column (collect the flow through).
- Wash column twice with 15ml of 10mM Tris pH7.5 and once with 10mM Tris pH 7.5 and 0.5M NaCl.
- 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.
- Wash column with 10mM Tris pH 8.0 until the pH of flow through is 8.0 (about 10ml).
- 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.
- 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