Coupling Antibody to CNBr Sepharose
1. Dialyze purified antibody against 0.2 M NaHCO3, pH 8.9. Use SpectraPor 2 (12-14 kDal cut off)
tubing and tubing clips. The volume of antibody solution should decrease during dialysis. Dialyze
overnight at 4°C in at least 100 volumes of buffer. Change buffer three times.
2. After dialysis, determine the protein concentration (BioRad Dye method). You will need 2-8
mg/ml final concentration. If needed, concentrate the antibody by Amicon or Centricon filtration.
Place antibody solution in 15 ml red cap tube on ice. Save a small amount (~10 µl) of antibody.
3. Wash 10 mls of packed Sepharose 4B with 50 mls ddH2O.
Wash procedure: Use sintered glass funnel and vacuum flask. Resuspend without suction. Wait ~1
minute. Apply vacuum until a dry cake is obtained. Do not over dry.
4. Wash with 50 mls 1M Na2CO3, pH 11.
5. Transfer packed Sepharose to a small glass flask and place on a rotating platform in a fume hood.
Add 10 mls of 1M Na2CO3, pH 11 to the flask, washing down walls of the flask in the process.
6. Dissolve 1 g of CNBr in 1 ml acetonitrile in 15 ml tube. Do in fume hood. CNBr is highly toxic!
7. While swirling the Sepharose suspension, add CNBr solution. Swirl for 10 minutes. Check pH
after addition of CNBr. Use ColorpHast pH paper (pH 7.5-14). Add 4 N NaOH to adjust the pH to
10.5-11. Add 0.5 ml at a time; 2-3 mls may be needed. Check and adjust pH every 2 minutes.
8. Transfer Sepharose to sintered glass funnel in vacuum flask in fume hood. Apply vacuum to remove
CNBr solution. Collect CNBr in 50 mls of 2% FeSO4-7H2O in vacuum flask.