Basic saturation binding experiments to determine dissociation constants ) assume that the concentration of free ligands is about the same (greater than 90%) as the concentration of ligands added. If not, the results could be invalid due to ligand depletion. A general rule of thumb is to use a starting concentration of protein well below (~10 times) the estimated , and ligand concentrations that bracket the by at least 10 times on either side. Many published experiments ignore this rule. This graphical aid simplifies experimental design by readily showing safe macromolecule/ligand concentrations in black and dangerous conditions in red. , , and all have the same units of concentration (nM in this Demonstration).