CNBr Activation of Agarose Beads:

  1. A chosen volume of agarose beads are washed with distilled water (> 4 bead volumes) and drained- but not dehydrated-on a sintered glass Buchner funnel attached to a vacuum filter flask. It is particularly important that any alcohols or glycol preservatives be completely removed from the beads at this stage, since they would otherwise compete with agarose hydroxyls (for CNBr) and result in lower levels of agarose activation.

    Re: Agarose bead concentration: the beads can have an agarose concentration between 1 and 10 % agarose.

  2. The washed beads should be free of any supernatant water but still moist and not beginning to dehydrate - particularly if they are < 4 % in concentration since dehydration can irreversibly change bead porosity.

    Note : Steps 3 - 8 & 12 MUST BE DONE IN THE HOOD. CNBr has the capability to release poisonous cyanide gas IF ACIDIFIED. AS A RESULT, IT IS IMPORTANT TO KEEP THE PH OF THE CNBr REACTION IN THE ALKALINE pH RANGE ( ideally pH 10.2 - 11.0). Keeping the reaction mixture cold is also important to obtaining maximum activation and avoiding premature hydrolysis of activated hydroxyl groups.

  3. The beads are then slurried with an equal volume of cold (20°C) distilled water and gently stirred in the hood.

  4. To control pH, a solution of NaOH should be prepared. The amount of NaOH required depends on the amount of CNBr added. For 5- 10 ml of agarose beads(and 1- 3 g CNBr), a 2 M solution should be prepared.  Note that the NaOH neutralizes the HBr formed when CN reacts with a hydroxyl group. For larger quantities of agarose beads (100- 200 ml and 20-30 g.of CNBr), a higher concentration of NaOH ( like 6M or 8M ) helps to minimize the total volume of the reaction mixture.

  5. The beads are then activated with CNBr by adding the required amount directly to the cold,stirred bead slurry( 50- 200 mg of CNBr/ml of beads; depending on level of activation desired.

  6. NaOH solution is IMMEDIATELY added so that the pH is adjusted to between pH 10.5 and pH 11 for the remainder of the reaction. As the activation proceeds the pH will be seen to fall and more NaOH solution will need to be added.

    Note : Cyanogen bromide (CNBr) is available from the Aldrich Division of Sigma-Aldrich( cat# C91492-25 g. for the 25 g. quantity; other quantities available).

  7. The reaction is usually complete in 6 - 12 minutes ( i.e. when the pH has stopped decreasing and no more NaOH is needed to keep it in the pH 10.5- 11.2 range.

  8. The activated beads are then washed with 10- 15 bead volumes of cold(10-20°C) 0.1 M NaHCO3 on a sintered glass Buchner funnel.

  9. The resultant agarose beads are now ready for coupling to a suitable protein ligand (i.e having at least one primary amine).
    1. The CNBr-activated beads are suspended in one volume of cold (10-20°C)0.1 M NaHCO3 and gently stirred.
    2. An appropriate amount of the protein to be coupled is also dissolved in one volume of 0.1 M NaHCO3 and thenadded to the beads with continued stirring.

      Note: There should be at least a 30 % excess of the protein (i.e milliequivalents of amine)to be coupled than calculated CNBr-activated sites ( in milliequivalents). For maximum coupling capacity, using as much as 20 times the desired amount of coupling ligand produces the best results.

  10. The ligand coupling step is allowed to proceed with continued stirring overnight at 5-10 °C.

  11. On a sintered glass funnel, the beads are then washed free of excess protein (i.e. ligand)and the excess ligand recovered as needed. The beads are washed sequentially with 2 bead volumes of 0.5 M NaCl, 0.1 M borate buffer, 1 M NaCl, 0.5M NaCl, 0.1 M NaOAc, 1 M NaCl, and finally with 0.5 M NaCl.

  12. Blocking any unreacted sites : The ligand-coupled beads are then resuspended in 2 bead volumes of 1 M NaCl,stirred gently, followed by addition of a 2:1 mole ratio of ethanolamine to CNBr used. The ethanolamine reaction is allowed to proceed for 1-2 hr.

  13. Final wash & storage: the beads should be washed with several bead volumes of 0.1 M saline until there is no odor of residual ethanolamine. The beads should be placed in a suitable preservative solution ( 0.03 % sodium azide or equivalent) and stored under refrigeration ( 4-10°C) until used.

    Note : The beads should NOT be frozen since non-crosslinked beads will be irreversibly damaged by freezing.

Reference(s):

  1. Z. Er-el, Biochem.Biophys. Res. Commun., 49, No.2, 383-386 (1972).
  2. R. Axen and S. Ernback, Eur. J. Biochem. 18, 351 ( 1971).
  3. J. Porath, Nature(London) 218, 834 (1968).