It could be an indication that your protein is suffering proteolysis during purification. Include a protease inhibitor cocktail in the extraction buffer, and keep everything cold during purification.

It's also possible that the bands you see are not the protein of interest, but are just some non-specific proteins that stuck to the Ni beads. The protein may not have been expressed, or it may have been expressed in an aggregated or insoluble form that does not bind to Ni resin.

Hi Llew,

do you perform purification protocol according to the nickel resin datasheet?

I think you might be using a high amount of nickel resin.

Non-specific proteins with histidine sequences (poly His sequence of the protein) may bind to the resin non-specifically, and this could be one of the reasons why you are getting two bands.

I don't have any idea why your enzyme is not active By ELISA.

With best regards.

Hamed Moslehi

Is the protein identity confirmed by Western blot?

Yes both bands confirmed to be protein of interest by western blot, so Im assuming cleavages is occurring at the smaller band ?

Some years ago I had an uncanny fragmentation of protein visible on SDS-PAGE. After puting the hints together, I made alternative sample preparation. The standart at 95 deg. C - 10 min. And two alternative in the presence of protease inhibitors. BUT at 45 deg. C and 70 deg. C. The lower temperature preparation resulted in a band of expected MW. The polypeptide got chemicaly cleaved during the heat treatment for sample preparation. Article Common Artifacts and Mistakes Made in Electrophoresis