To fit a binding curve:

1. Start from a table where X is the concentration of ligand in nM or pM and Y is specific binding. This can either be the original data table or a results table.

2. Click Analyze.

3. From the curves and regression choices, choose Nonlinear regression.

4. On the nonlinear regression dialog, choose One site binding. Leave everything else at the default settings. The one-site equation is

5. Press ok. You'll see the table of results. Bmax is expressed in the same units as the Y values (commonly cpm, sites/cell or fmol/mg). Kd is expressed in the same units as the X values, usually nM or pM. If the Kd is low, the affinity is high.

To get useful results with this approach, you will need high quality data and at least ten data points including some well above the ligand Kd.   

Determining Kd and Bmax for two classes of binding sites

If the radioligand binds to two classes of binding sites, fit the specific binding data to the two-site binding equation (found in Prism's list of built-in equations). This equation simply sums the binding to two sites, each with its own Bmax and Kd .

This equation assumes that the radioligand binds to two independent noninteracting binding sites, and that the binding to each site follows the law of mass action. To compare the one-site and two-site fits, see Comparing the fits of two models

You will only get meaningful results from a two-site fit if you have ten or more (preferably a lot more) data points spaced over a wide range of radioligand concentrations. Be sure that you measure binding at radioligand concentrations below the high-affinity Kd and above the low affinity Kd.