Generation of an Affinity Column with Sepharose

Biochemistry, Molecular Biology, and Cell Biology Protocols  >> Generation of an Affinity Column

1. Take 10 ml of Sepharose 2B, and wash several times with water by centrifugation.  Do not centrifuge too fast as it may break the Sepharose beads.  Centrifuge just fast enough to pellet the beads.
2. Pour the washed Sepharose into a small beaker (roughly 50-100 ml) containing an equal volume of water, and stir gently with a stir bar in the fume hood.
3. Take out cyanogen bromide approximately half an hour prior to needing to use it.  Please note that cyanogen bromide is extremely toxic and should be opened in the fume hood.  Also, any solution that has come into contact with cyanogen bromide should be used in the fume hood.   Weigh an empty 15 ml Falcon tube with a sensitive analytical balance that can record 4 decimal places, and tare the balance.  Leave a note on the balance saying that it is in use.  In the fume hood, use a spatula to transfer a little bit (should be approximately 0.19 g) into the Falcon tube.  Leave the spatula in the fume hood until the end of the procedure when you rinse the spatula in the fume hood.  Go to the balance to check the weight of the cyanogen bromide.  If there is not enough or too much, go back to the fume hood to adjust accordingly.  Remove the note from the balance once you are done.
4. Have a bottle of 0.2 M NaOH ready in the fume hood, and add a drop of NaOH into the beaker with the Sepharose, with the stir bar stirring, and the electrode of the pH meter in the solution. (The electrode should first be rinsed with water.) The pH should reach a value between 10-11.  If not, add more NaOH until that pH is reached.
5. Add the cyanogen bromide to the beaker.  Keep stirring.  Maintain the pH of the solution between 10-11 by adding 0.2 M NaOH as the pH drops. Continue for 30 minutes or slightly longer until the pH stabilizes reasonably well.
6. In the fume hood, transfer the solution to a 50 ml Falcon tube, add some cold 20 mM sodium borate, pH 8.4, and cap the solution tightly.  Centrifuge just quickly enough to pellet the beads.  In the fume hood, open the tube, remove the solution with a pipet and into the fume hood sink or a waste beaker in the fume hood.  Repeat the washing 3 more times. 
7. After the last wash, remove the sodium borate, add approximately 16 mg of concentrated antibody or other protein dialyzed into 20 mM sodium borate, pH 8.4, to the beads.  It may be more convenient to transfer the beads and antibodies to a 15 ml Falcon tube at this step if the volume fits.
8. Rock, rotate, or mix the bead-protein solution at 4 degrees C for 4 hours.
9. Wash the glassware or other laboratory equipment that has touched cyanogen bromide in the fume hood.
10. After 4 hours, check the A280 to ensure that it has decreased, and most of the antibody or protein has bound.
11. 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.
12. Store the affinity column at 4 degrees C in TBS without glycine.
13. The affinity column can be used in small volumes.  For more information on purification procedures, please see Immunoaffinity Chromatography Protocol.

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