Do not ask two quetions in one question and do not answer your own question with a new. This wil not help you and people get annoyed.

First question: you have to fit and calculate ka and kd to compare two sensorgrams. But really first, make your sensorgrams better. These sensorgrams will give you wrong answers. --> Too much bulk and shape is not 1:1.

Second question: without knowing the molecular weight of the ligand and analyte I don't know what the immobilization level should be. And I am confused. Do you the experiments yourself or not?

So you have learned to give all the information you have to get the best result/answer.

kind regards

Arnoud

Dear sir,

I didn't do the experiment myself but sent my protein and analytes for the service. It was failed to determine the kinetics because of the NSB, and the service was ended. I couldn't contact them to ask, and it is quite difficult for me in the first step to understand this method by reading on my own. Now I am trying to understand the result report with only the sensorgrams and figure out what is wrong. There is one beginning step I doubt it will affect the result is that my ligand pI is estimated 4.74 which is too close to their used dilution buffer pH for immobilization (SA, pH5.0) for amine coupling. My protein is truly turned turbid in that buffer.

Otherwise, I am reading to understand the NSB troubleshooting, which BSA using and adding Tween-20 didn't help in their re-run. I was confused because my analyte pI>9, the running buffer pH~7, perhaps caused NSB with the sensor or the protein was not in the right conformation for correct binding.

Anyway, please forgive me for my unclear and confusing way of asking. There are still too many basic terms I need to understand it better before asking.

Thank you so much for your time and effort to help me.