For the immobilization process, you can begin by calculating the amount of RU of protein needed for an Rmax for peptide binding of ~30. This depends on the molecular weights of the protein and peptide and the valence (usually 1 ). Then, when you do the pH scouting experiment for immobilization, choose the highest pH that gives the necessary RU, since most proteins are more stable at neutral pH than at the acidic pHs usually used for the immobilization step.

The running buffer pH should be chosen to be physiologically relevant for the protein. Phosphate buffer or HEPES are often used for the so-called physiological pH of ~7.4. The buffer should contain at least 0.05% Tween-20 detergent. You can buy prepared running buffers from Cytiva, or use what they sell as a guide for making your own to save money.

The concentrations of analyte should span above and below the Kd, about 10-fold either way. Since you don't know the Kd to start, you could begin with a prelimimary experiment testing a range of concentrations with a set of 3- to 10-fold serial dilutions. Once you find the suitable range at which you get about half-maximal saturation, do a second experiment with a narrower range from 1/10 to 10 times that concentration.