Lectin Gel Procedure

USE

1. The reaction may be carried out in a test tube (which will require centrifugation for washing steps) or in a small column (either a glass pipette or a plastic mini-column).  Gels may be run at room temperature or in a cold room.  Elevated temperatures should be avoided.

2. Wash gel with 10 times the gel volume using the appropriate Buffer.  Many proteins will require different buffers, pH’s, and ionic conditions for binding.  Many will also require the addition of specific ions to insure binding.  These conditions must be determined, or discovered experimentally by the researcher.

3. Apply the sample and wash the unbound material from column with the Buffer of choice.  DO NOT OVERWASH!!  The affinity for the carbohydrate/lectin depends on the binding constant of the protein.  Extensive washing may elute the protein to be purified if the binding constant is low.

4. Elute bound material using the appropriate carbohydrate in the Buffer of choice.  Collect small
samples.  Unless the optimal carbohydrate concentration has been previously determined it is
recommended to start with a 0.1M‑0.2M solution.

Gel Regeneration

1. After elution, wash the gel with 10 times the gel volume using 1.0-1.4 M NaCl in distilled water.  Re-
equilibrate the gel by washing with 50 times the gel volume using the appropriate Buffer.  Store
refrigerated with 0.1% sodium azide as a preservative.  DO NOT store the gel in a high salt
concentration solution. DO NOT FREEZE.