To set up the Kinetic assay for RBPR2 and STRA6 peptides serially dilute the mouse RBPR2, mutant RBPR2 and STRA6 peptides in running buffer. Open the control software and select the Kinetic SOAR Affinity wizard. In the injection sequence dialogue, select Flow path 2.1 with Flow cell 2 as active and Flow cell 1 as reference.
In the setup dialogue, check the run startup cycles option, input the solution as buffer, specify the number of cycles and click next. Now go to the injection parameters dialogue and enter sample parameters such as contact time of 120 seconds, flow rate of 30 microliters per minute, and dissociation time of 300 seconds. For regeneration parameters, set the regeneration solution to glycine hydrochloric acid with a contact time of 30 seconds and a flow rate of 30 microliters per minute, then click next.
In the samples dialogue enter the peptides names, molecular weights, and concentrations, then click next. In the rack position dialogue assign the diluted peptides as individual 120 microliter samples with various concentrations from one to 100 micromolar to the designated positions on the rack. Click eject rack and insert all the vials.
Afterward, click next and then in the prepare run protocol dialogue click start. Enter a file name and click save. Now in the software, click the start menu and select a valuation.
Open the file then from the dropdown selection menu choose FC-2.1 to analyze the reference cell blank subtracted active sensorgram for the association and dissociation phases of msRBP4, mutant msRBPR2, and msSTRA6 peptides. Next, click the dropdown Kinetics or Affinity button and choose surface bound. In the select curves dialogue, check include column in the table, and click next.
Now go to the select data dialogue, view the blank subtracted sensorgrams. Click Kinetics and Affinity. Keep the default parameters and click fit for curve fitting.
Inspect the report tab for equilibrium dissociation constant Kd, association rate Ka or Kon and dissociation rate Kd or Koff. Save the Kinetics data sensorgram in text tab to limited format and use the values to plot graphs.
