To troubleshoot and optimize your SPR experiment:

1. High Kd in positive analyte control: Optimize ligand immobilization conditions to improve binding.

2. Non-specific binding: Optimize blocking agents and injection conditions to minimize non-specific interactions.

3. Ligand density: Evaluate different ligand concentrations for optimal binding.

4. Running buffer: Consider alternative buffers or modify composition to reduce non-specific binding.

5. Conduct systematic optimization experiments for ligand density, injection conditions, regeneration, and running buffer.

6. Include appropriate negative controls to validate specific binding and minimize non-specific effects.

Thank you for your suggestions. I wonder if non-specific binding occurred because my analyte pI is too high and positively charged in the running buffer pH < analyte pI. We have the negative control of the analyte and also the reference ligand, the KD still can't be estimated.

Though KD can't be estimated, if RU of one analyte is higher than another (binding/stability: 50/33 vs 80/52), can I suppose that one may bind stronger than the RU-lower one?

"if RU of one analyte is higher than another (binding/stability: 50/33 vs 80/52), can I suppose that one may bind stronger than the RU-lower one?"

No! - there are many parameters contributing to the response level of a ligand - analyte combination. Only proper fitting of the whole binding curves can give a real answer to the bidning/affinity question.

You can rank the binding on dissociation rate constants provided that the dissociation goes back to baseline (no re-binding or sticking non-specific bound compounds)

Please consult www.sprpages.nl and the sensorgram tutorial