Negative response final after ligand immobilization on SA chip

Hi to all,

I am pretty new to SPR and now have been facing an issue regarding ligand immobilization on the SA chip.
I did immobilize a ligand (20 nM biotinylated recombinant protein diluted in HBS-P+ buffer) on a flow cell of the chip with the aim of immobilization level at 200 RU by following default immobilization protocol in wizard template.
As result, I got a response bound of 190.6 RU but the response final was -213.2 RU.

What does this result mean? How should I troubleshoot this issue?

Any suggestion would be appreciated in advance.

Hi,

Can you post the sensorgram so we can see what happend?

Arnoud

Hi Arnoud,
Thanks for your response. I did manage to solve the issue already by increasing the ligand concentration. The actual ligand immobilization level was at response final 150 RU.

However, once the protein analyte concentrations were flown on reference and active flow cells, the negative response after subtraction was observed.
I speculate that this may due to the insufficient immobilized ligand molecules. This is because I can previously see the specific interaction of this ligand and analyte (positive response) when the immobilized ligand level of 1,500 RU was used.

My question is how can I immobilize more ligand molecules on the SA chip surface without perturbing the pre-immobilized one?

Any suggestions are welcome and appreciated.

Hi Joyce,
If you have negative responses after subtracting the reference you shoud check if the analyte is not binding to the reference channel more than the active channel. You may want to block the reference with D-biotin and see if this will lower the non-specific binding to the reference.
Adding more ligand to the active channel is always possible without blocking the 'old' ones. You can add ligand until all free positions are filled. But use the lowest ligand density to avoid non 1:1 interactions.

Kind regards
Arnoud