Hi Mehmet,

If the system is under equilibrium and there is no lateral diffusion (or it can be neglected), then the fluorescence recover rate after photobleaching is equal to the dissociation rate of your labelled molecule. This is quite intuitive since the recovery is equivalent to dissociation of the bleached molecules, however, it's fair easy to show this more rigorously with equations. I think that's what most people assume when using FRAP to determine koff  values.

If that's also your case, then the dissociation rate determined from FRAP is actually directly related to association rate. Assume your reaction is A+BC, with forward and backward rates being kon, koff respectively. Since the system is under euqilibrium, then you can calculate kon from the concentrations of each species and koff by 'kon[A][B]=koff[C]'. The tricky part is, those concentration values are the equilibrium concentrations, not the concentrations you begin your experiment with. So you'll need to estimate or calculate those equilibrium concentrations. I'm not sure what your system is, but in many cases the equilibrium concentrations are easy to estimate once you know the reaction mechanism and initial values.

Hope this helps,

Zheng