Agarose Beads Column Packing Procedure

  1. Washing the beads: BioScience Agarose Beads are supplied fully hydrated and contain either 0.03% sodium azide or 17% ethanol as a preservative.

    1. In either case, the gel beads shold be placed in at least 3 bead volumes of distilled, deionized, or reverse-osmosis (RO) treated water and gently stirred for 10 minutes.

    2. The bead slurry should then be passed through a filter which will retain the beads. A sintered glass filter in a Buchner funnel attached to a vacuum flask is quickest. Note : do not overdry the beads. If the the surface of the beads begins turning an opaque white, you are beginning to dehydrate the bead surface and low concentration (< 4%) beads can irreversibly dehydrate under these conditions.

  2. Equilibration in starting buffer: Place the filtered beads in 3 bead volumes of the desired application buffer and stir gently for about 10 minutes.

  3. Degassing the bead/buffer slurry: Place the beads slurry back in the vacuum filter flask and stopper the top before applying a vaccuum for about 5 minutes. Occasional swirling of the container will help to fully degas the slurry. Note do not use a stir bar in preference to manual swirling. (Save the degassed buffer for a later step).

  4. Filling the column with buffer: Fill the column about 1/3rd full with degassed, starting buffer and dislodge any air bubbles that might be trapped in the column end pieces or bed support membrane. Use a combination of tapping the column and draining the buffer through the exit stopcock to accomplish this. Close the stopcock and replenish the degassed to 1/3rd the column height.

  5. Filling the column with beads: Fill the column with a filling funnel and reservoir and add the 50:50 bead/buffer slurry (a convenient pouring concentration if well mixed). Allow the beads to settle until there is a bed height of about 4 cm.

    1. Open the stopcock and allow buffer to flow out until a stable , well-packed bed of beads has formed.

  6. Connecting the column to the pump: Connect the top of the column or flow adapter (recommended for SEC, IEC, HIC but is less critical for affinity chromatography, AIC) to the pump or Marriotte flask using some appropriate, non-leaching plastic tubing. Fill the tubing and pump in the standard manner to insure that they are free of air bubbles.

    1. If a column reservoir is used, drain any excess buffer from it and attach the flow adapter to the column.

    2. Pump or drain at least 4-5 bead bed volumes of buffer though the column at a slightly higher flow rate than will be used during the planned chromatography.

  7. Adjusting for bead bed volume changes: Turn off the pump and close the column outlet. Reposition the flow adapter until it is just making contact with the top of the gel bed.

  8. Final equilibration with starting buffer: After the gel bed had equilibrated for about 10 minutes with the starting buffer, the column is ready for sample application.

  9. Sample application: The liquid sample is applied as a thin, uniform layer to the top of the gel bed and the buffer flow ( either by gravity, using a Marriotte flask or using a pump) is begun.

  10. Chromatography: Buffer flow is continued until the desired separation of sample components has been achieved and each component has both eluted from the column and been collected. Quantitation of each component can be done either continuously, during elution, or after collection.