Generation of an Affinity Column with Sepharose

  1. Take 10 ml Sepharose 2B, and wash several times with water by centrifugation. Do not centrifuge too fast as it can break Sepharose beads. Centrifuge just fast enough to pellet beads.
  2. Pour into glasses washed Sepharose small (approximately 50-100 ml) containing the same volume of water, and stir gently with a stir bar in a fume hood.
  3. Remove cyanogen bromide about half an hour before needing to use it. Please note that the cyanogen bromide is highly toxic and must be opened in a fume hood. Also, any solution that has come into contact with cyanogen bromide should be used in a fume hood. Weigh empty 15 ml Falcon tube with a sensitive analytical balance to record fourth decimal place, and tare the balance. Left a note on the balance said that it is used. In a fume hood, using a spatula to transfer the bit (to be approximately 0.19 g) to the Falcon tube. Let spatula in a fume hood until the end of the procedure when you rinse spatula in a fume hood. Go to the balance to check the weight of cyanogen bromide. If there is not enough or too much, go back to the hood to adjust accordingly. Clear records of the balance after you have finished.

Generation of an Affinity Column with Sepharose

  1. Having a bottle of 0.2 M NaOH prepared in a fume hood, and add a drop of NaOH into a glass with Sepharose, with a stirring rod, and a pH meter electrode in the solution. (First electrodes should be rinsed with water.) PH should reach a value of between 10-11. If not, add NaOH until the pH reached.
  2. Add cyanogen bromide into a beaker. Stirring constantly. Maintaining the pH between 10-11 by adding 0.2 M NaOH as pH drops. Continue for 30 minutes or a little longer until a stable pH pretty good.
  3. In a fume hood, transfer the solution to 50 ml Falcon tube, add some cold 20 mM sodium borate, pH 8.4, and cap tightly solution. Centrifuge just fast enough to pellet beads. In a fume hood, open the jar, remove the solution with a pipette and to sink a fume hood or glass waste in a fume hood. Repeat washing three times more.
  4. After the last wash, remove sodium borate, adding about 16 mg of antibody or other protein concentrated dialyzed in 20 mM sodium borate, pH 8.4, with beads. It might be easier to transfer beads and antibody for 15 ml Falcon tube at this step if suitable volume.
  5. Rock, rotate, or a mixture of bead-protein solution at 4 degrees C for 4 hours.
  6. Wash laboratory glassware or other equipment that has touched cyanogen bromide in a fume hood.
  7. After 4 hours, check the A280 to ensure that it has declined, and most of the antibody or protein has been bound.
  8. Stop the coupling reaction by washing the Sepharose beads twice in TBS (20 mM Tris, pH 7.4, 150 mM NaCl) with 50 mM glycine by centrifugation.
  9. Store affinity column at 4 degrees C in TBS without glycine.
  10. The affinity column can be used in small volumes. For more information about the purification procedure, please see immunoaffinity Chromatography Protocol.

Generation of an Affinity Column with Sepharose

Leave a Reply

Your email address will not be published. Required fields are marked *