In many experimental situations, you can assume that a very small fraction of the ligand binds to receptors (or to nonspecific sites). In these situations, you can also assume that the free concentration of ligand is approximately equal to the concentration you added. This assumption vastly simplifies the analysis of binding experiments, and the standard analysis methods depend on this assumption.
In other situations, a large fraction of the ligand binds to the receptors (or binds nonspecifically). This means that the concentration of ligand free in the solution does not equal the concentration you added. The discrepancy is not the same in all tubes or at all times. The free ligand concentration is depleted by binding.
Many investigators use this rule of thumb: If less than 10% of the ligand binds, don't worry about ligand depletion; if more than 10% of the ligand binds, you have three choices:
•Change the experimental conditions. Increase the reaction volume without changing the amount of tissue. The problem with this approach is that it requires more radioligand, which is usually very expensive.
•Measure the free concentration of ligand in every tube. This is possible if you use centrifugation or equilibrium dialysis, but is quite difficult if you use vacuum filtration to remove free radioligand.
•Use analysis techniques that adjust for the difference between the concentration of added ligand and the concentration of free ligand. Prism includes such models for analyzing saturation and competition data. These special analyses only work with radioactive ligands, so the assessment of added ligand and bound ligand are in the same counts-per-minute units. These methods don't work with fluorescent ligands.