Saturation Binding of Ligands to Proteins

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).


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The equation used is
[1] K. B. Hall and J. K. Kranz, "Nitrocellulose Filter Binding for Determination of Dissociation Constants," Methods in Molecular Biology, 118, 1999 pp. 105–114.
[2] H. Motulsky, "GraphPad Prism Version 4.0 Statistics Guide," La Jolla, CA: GraphPad Software, Inc., 2005.
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