I am trying to use FRAP to measure the binding kinetics of two proteins. A is an immobile protein, which could be bound with B (mobile protein and with fluorescent tag). I photobleach one region with A and B bound, then wait for the recovery and measure the time course of FRAP. After repeating it under different concentration of B, I try to extract the on/off rate of B binding A by using the following equation:
My question is, can I do experiments like what I describe? Any criticism and concerns about the experiment design? thanks!!!