GraphPad Curve Fitting Guide

Key concepts: Saturation binding

Key concepts: Saturation binding

Previous topic Next topic No expanding text in this topic  

Key concepts: Saturation binding

Previous topic Next topic JavaScript is required for expanding text JavaScript is required for the print function Mail us feedback on this topic!  

What is saturation binding?

In a saturation binding experiment, you vary the concentration of radioligand and measure binding at equilibrium. The goal is to determine the Kd (ligand concentration that binds to half the receptor sites at equilibrium) and Bmax (maximum number of binding sites).

Total, nonspecific and specific binding

The ligand binds not only to receptors sites, but also to nonspecific sites. There are three approaches to dealing with nonspecific binding.

Subtract off the nonspecific, and analyze only the specific binding.

Analyze the total binding only, inferring the amount of nonspecific binding from the shape of the total binding curve.

Globally fit total and nonspecific binding together.

We recommend the third approach (global fitting of total and nonspecific). The problem with fitting specific binding is that you have to make some assumptions in order to subtract nonspecific from total, and the resulting values that you fit aren't really data. When possible, we suggest that you fit the data you actually collect, and avoid creating derived data sets (specific binding, in this case).

Fitting total binding only requires less data, so saves experimental time and money. But most people feel unconformable defining nonspecific binding purely from the shape of a binding curve, without experimentally measuring nonspecific binding. One advantage of fitting total binding only is that equations have been derived for fitting such data, even when a substantial fraction of the ligand binds, resulting in ligand depletion (free concentration substantially less than the added concentration).

One-vs. two sites

Prism offers models for fitting one or two sites. You can use choices in the Compare tab to compare the two fits. When comparing the fits to the one- and two-site models, use common sense as well as statistics. Don't accept a two site model, if one of the sites is only a tiny fraction of the total, or if its Kd is outside the range of radioligand concentrations you used in the experiment.