I agree with the two earlier posts, your protein is probably not in the native conformation, and hence no biological activity. There is also a chance that the high level of imidazole used to elute the protein off the affinity column may inhibit the activity of your protein. You can test this possibility by adding some of your eluted protein to your positive control.
Not affiliated with any company. Mention of a specifc product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.