Procedures for Coupling Ligands to Agarose Beads

A) Coupling an Amine-containing Ligand to the Aldehyde Moiety of a Glyoxylated Agarose Bead:

Coupling an Amine-containing Ligand to the Aldehyde Moiety of a Glyoxylated Agarose Bead

The aldehyde groups of Glyoxal Agarose Beads react with ligands containing primary amine groups to form Schiff Base Intermediates, which are selectively reduced by cyanoborohydride. Sodium borohydride should not be used for this procedure because it reduces both the unreacted aldehyde and Schiff Base groups thereby lowering maximum coupling capacity. The higher the pH of the buffer used in the reaction, the greater the rate and extent of Schiff Base formation and ligand coupling. The resultant ligands formed are much more stable and less prone to leaching than those coupled using CNBr (cyanogen bromide).

Procedure:

  1. Determine the amount of ligand you want to immobilize:
    1. The amount of ligand required will depend on how much "target" substance you want to isolate in one use of the affinity beads to be prepared.

    2. Estimate the binding ratio of ligand to target substance (or assume it's 1:1)

    3. From (a) and (b) above, you now know the number of µmoles or µ equivalents of ligand to be bound to the beads.

    4. Glyoxal agarose beads contain at least 20µ equivalents of aldhyde groups per ml of beads. Calculate the volume of beads required to bind the desired amount of ligand.

  2. Preparing the Schiff-Base Reduction (SBR) Solution:
    1. Using the Sigma Coupling Buffer (C 4187): This is highly recommended since this ready-to-use buffer minimizes the need to inventory the concentrated solutions of cyanoborohydride.

    2. Preparing your own SBR Solution: Follow the steps outlined at the end of this procedure in "Stock SBR Solution Preparation". Note: You have a choice of only two glyoxal-compatible buffers: phosphate (P) and borate (B). No amine-containing buffers should be used !

  3. Preparing the Glyoxal Agarose Beads: Filter a slurry of the glyoxal beads on a sintered glass filter or equivalent and weigh out the amount of beads required (1d above). Equilibrate the beads in 3 volumes (1g. beads = 1ml) of alkaline buffer (P or B) for 15 minutes.

  4. Equilibrating the beads: Filter the beads and add them to 2 bead volumes of fresh alkaline buffer (P or B). Stir for 15 minutes.

  5. Add the ligand as a dilute solution: Dissolve the ligand in water, saline, or alkaline buffer (Por B) and add it to the beads with stirring. Note: no amine-containing buffers should be present in the ligand soulution.

  6. Allow coupling reaction to go to completion: Allow the reaction to proceed for at least 2 hr. Filter the beads and resuspend them in 2 bead volumes of SBR Solution.

  7. Block unreacted glyoxal sites: Mix 20 µl of ethanolamine for every ml of glyoxal beads with 5 ml of water and add the resultant solution to the ligand-coupled bead slurry and allow the reaction to proceed for 1 hr. Then wash the beads on the filter funnel with at least 5 volumes of water or buffer (but not coupling buffer).

  8. Storage: The ligand-coupled glyoxal beads should be stored in a preservative-containing buffer which is suitable for the ligand.

Alkaline Buffer and SBR Stock Solution Preparation:

  1. Alkaline buffer: 0.2 M disodium phosphate ( Sigma # S-9290) or 0.2 M sodium borate (Sigma # S-9640) solutions should be prepared. Dissolve the required amount of either salt in water. Note: The sodium borate is available as the sodium tetraborate decahydrate ( 76.2 g/L) as well as the tetrahydrate or metaborate salts.

  2. SBR stock solution: Prepare a 2 M solution of sodium cyano-borohydride - in the hood. Dissolve 6.3 g of sodium cyanoborohydride in 50 ml of water. It is best to let this solution stand overnight before use to decompose any residual sodium borohydride which might be present.

  3. Schiff-Base Reduction (SBR) Solution Preparation : dd 1 ml of the SBR stock solution (B above) to 10 ml of a suitable alkaline buffer (A above) to form the SBR solution (0.2 M in CNBH4). Because this buffer is more alkaline than the “Sigma Coupling Buffer”, it maximizes the rate and extent of intermediate Schiff Base formation.

    Re: Safety:
    1. Cyanoborohydride solutions should NEVER BE ACIDIFIED (i.e. pH reduced below 7.O).

    2. Perspecitve : Cyanogen bromide solutions have been used routinely for more than 40 years to produce cyanogen bromide activated agarose beads. All of the same safety precautions necessary for CNBr activation also apply for cyanoborohydride reduction.

    3. Always use unbreakable containers to minimize the likelihood of spills.

References:

  1. Shainoff, J.R., "Zonal Immobilization of Proteins ", Biochem and Biophys. Res. Commun. 1980, 95, 690.

  2. Shainoff, J.R. "Glyoxal Agarose" , U.S. Patent 4,275,196.

  3. Guisan, J.M et. al, "Immobiliztion of enzymes on glyoxal agarose", Methods in Biotechnology, 277-287 ( 1997).

  4. Hermanson, G.T. et. al, "Immobilized affinity ligand techniques", pg. 69-75, Academic Press, Inc. , San Diego, CA (1992).

  5. Hearn, M et. al, J. Chromatog. 185, 453 (1979).

  6. Lane, C.F., "Sodium Cyanoborohydride- a highly selective reducing agent for organic functional groups ", Synthesis, 3, 135-146 ( 1975).

  7. Mosbach, Klaus, in "Methods in Enzymology" , Vol. XLIV: Immobilized Enzymes, 1976
    Academic Press, NY.

B) Coupling a Carboxyl-containing Ligand to the Amine Moiety of an Aminoethylated Agarose Bead:

Coupling a Carboxyl-containing Ligand to the Amine Moiety of an Aminoethylated Agarose Bead

Procedure:

  1. Wash the required amount of aminoethyl (AE) agarose beads in 3-4 bead volumes of
    0.5 M NaCL.

  2. For every 25 ml of AE agarose beads, prepare 1- 1.5 mM solution of EEDQ in 25 ml of ethanol. Use water instead of ethanol for the water soluble CDi's .

  3. Add 0.5-1.5 mM of the ligand in 50% ethanolic EEDQ to the AE beads. The ligand can be dissolved in water for water-soluble CDi's . The volume of the ligand solution should not exceed 1.0 ml/ 2.5 ml of AE beads to avoid excess dilution.

  4. Agitate preferable by rocking or swirling. Note: a typical magnetic stir bar or overhead
    stirrer-impeller will tend to damage the gel beads - particularly at high speed. Stir or mix overnight at room temperature ( 20-25°C).

  5. Wash the AE beads with 50% ethanol for EEDQ, otherwise with water for other CDi's, until excess reagents or byproducts are eliminated.

  6. Repeat washing with 3-4 bead volumes of 1 M NaCL followed by a rinse with DI or DO water.

  7. The ligand is now coupled to the AE beads via an amide bond and the beads may be either used for affinity chromatography or stored in 0.03% sodium azide ( 4-10°C).

Coupling Reagent Nomenclature and Acronyms:

  1. A water soluble carbodiimide (CDi) is preferable:
    1. CMCi : 1-cyclohexyl-3(2-morpholinoethyl) carbodiimide-metho-p-toluene sulfonate

    2. or EDCi: 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride.

  2. An Alchohol/water soluble CDi is next best: EEDQ : (N-ethoxycarbonyl-2-ethoxy-1,2- dihydroquinoline)

  3. Less desirable, unless hydrophobic solvents are used: DCC : dicyclohexyl carbodiimide.