Hi Shradra,

maybe you need to optimize and adjust the pH of your binding and elution buffers? Have you checked this?

Best,

Murat

Remove imidazole concentration and determine if your protein of interest binds to the metal ions, if so, run a slow imidazole gradient. If not (and assuming all regular buffer conditions are met), then the his tag is not accessible.

Dear Shradha Surin

which kind of mutation do you performed?

If the mutation is located outside the histidine tag and the protein is not unfolded by the mutation, theoretically there are no reason why it has to affect the protein binding a Ni-Nta coloumn. It seems that the mutation induce protein unfolding, therefore your protein aggregates (as happen some times when membrane proteins are fused with MBP) and therefore the his-tag are buried inside the aggregates and not able to bind the coloumn.

The overexpression level and solubility in the gel of the WT and mutant protein is comparable?

best regards

Manuele