CNBr-Sepharose reacts with amino groups. I don't think it reacts with hydroxyl groups (CNBr itself reacts with hydroxyl groups, which is how it activates the resin). So your compound probably wasn't immobilized, unless it has amino groups.

When immobilizing a compound on a column, it is a good idea to have a linker to provide some spacing between the resin and the compound, so that a protein can bind.

You might be able to immobilize your hydroxylated compound by first oxidizing it to an aldehyde or carboxylic acid, then using the appropriate resin for immobilizing those functional groups. Such resins are available here:

https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/protein-purification/protein-immobilization.html

It is important to remember that part of the small molecule will be changed by the immobilization, and if this part is crucial for protein binding, the protein may no longer bind.

A convenient way to tell whether the immobilization has worked is to measure the amount of ligand in the supernatant before and after coupling.

Dear Mr. Saphiro,

thank you very much for your kindly explanation.

At the first time I thought that epoxy-sepharose beads will be more suitable to immobilize compound with -OH groups. But since the epoxy beads was not available yet in our lab, we tried first with CNBr beads. We found some paper about immobilization of -OH group (instead of -NH2) on CNBr beads, such as:

http://cancerpreventionresearch.aacrjournals.org/content/7/4/466.long (curcumin)

http://www.sciencedirect.com/science/article/pii/S0304383515000749 (rottlerin)

http://cancerres.aacrjournals.org/content/68/14/6021 (myricetin)

but now my lab try to get some epoxy-sepharose beads, will that be more suitable?

Thank you very much for your help.

best regards,

Yonika

Epoxy-activated Sepharose is intended for immobilization through OH groups of sugars and other carbohydrates. This product has a spacer arm, which is desirable for reducing steric hindrance due to proximity to the resin surface.

http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-us/products/AlternativeProductStructure_17313/17048001