Could be the pH, the buffer components... are you sure you are using the right ligand? Is it freshly prepared? Does the receptor need post-translational modifications? Is it correctly folded? Could there be a competitor in your solution?

Dear Anna

Thank you for your response.  The pH, buffer, and ligand are Ok. But I don't know about post-translation modifications or competitors. How could I measure them?

I'm afraid I don't know about them either, but a few things to think about are -is the protein you are working with purified from the natural source? - is it heterologously synthesised (which could lead to missing modifications, mis-folding, missing partner protein necessary for function)? -is the protein getting too old? -was it stored in optimal conditions?

Also, are you 100% sure you are using the right method to quatify a positive response? You have not provided any experimental details in your question... Are you following a published protocol?

I hope this helps,

-Anna