Fluorescence polarization experiments are very easy to set up and perform, so you might as well just do the experiment to find out the actual Kd.

First, you must decide on the concentration of the FAM-labeled aptamer, which will be fixed. You want its concentration to be substantially below the Kd of the interaction, if possible (see below), but also high enough to give a good signal. You can begin by varying the aptamer concentration and measuring the fluorescence intensity. A decent fluorescence intensity signal is at least 20 times the background from zero concentration. You can settle for less, if necessary.

Once you have chosen the aptamer concentration, all you have to do is mix equal volumes of 2X aptamer and a set of serial dilutions of the protein. Two-fold serial dilutions of the protein will work well.

As an example, suppose you add 10 nM aptamer (5 nM final). You could start with 2 µM protein and prepare a set of 2-fold serial dilutions down to about ~2 nM, plus zero, for a total of 12 samples. After mixing the samples, read the polarization at various time intervals (e.g. 5, 10, 20, 40 minutes) to see whether there is any time-dependence of the Kd.

The Kd can be calculated from the polarization measurements using a variation of the Hill equation (without cooperativity) and a nonlinear regression computer program.

P = Po + Pmax[L]/(Kd + [L]).

P is polarization at ligand concentration [L], Po is polarization when [L]=0, Pmax is polarization when all the aptamer is bound to protein). You would enter the values of P and [L] for each ligand concentration and let the computer find the best-fit values of Po, Pmax and Kd (or you could set the measured value of Po as a constant).

This equation is only correct as log as the Kd is substantially higher than the aptamer concentration, because of the assumption that the free and total protein concentrations are equal. If the assumption is false, you can use the so-called Morrison equation instead, which does not make that assumption.

Thank you so much for all this information, this is very useful for me!

Sincerely

Ana