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Radioligand Binding Assays Explained: Kd, Ki and Bmax for Peptide Receptors
Before a peptide can do anything to a cell, it has to bind. Radioligand binding assays are the oldest and most direct way to measure that step, and they produce three numbers that every receptor pharmacologist uses: Kd, Bmax and Ki.
Key takeaways
- A saturation assay measures how much labeled ligand binds as its concentration rises, giving Kd and Bmax.
- A competition assay measures how well an unlabeled compound displaces the label, giving IC50 and then Ki.
- Nonspecific binding must be measured and subtracted.
- Peptides stick to plastic, which can distort the numbers.
- Binding tells you about affinity, not about whether the compound activates the receptor.
The basic idea
Prepare cell membranes or intact cells that carry the receptor. Add a radioactive version of a ligand at known concentrations. After the mixture reaches equilibrium, separate what is bound from what is free, often by rapid filtration, and count the radioactivity on the filter.
Saturation: Kd and Bmax
In a saturation assay, you increase the concentration of labeled ligand and measure binding at each step. The curve rises and then flattens. The concentration at which half the receptors are occupied is the Kd, the dissociation constant. A smaller Kd means tighter binding. The plateau is Bmax, the total number of receptors in the sample.
To get specific binding, a second set of tubes includes a large excess of unlabeled ligand to block the real receptors. Whatever counts remain are nonspecific, and are subtracted.
Competition: IC50 and Ki
Most researchers want to know how a new compound compares with a known one. In a competition assay, a fixed amount of labeled ligand is combined with increasing concentrations of the unlabeled compound. The more it competes, the less label stays bound. The concentration that cuts binding in half is the IC50.
IC50 depends on how much label you used, so it is converted to Ki with the Cheng-Prusoff equation: Ki equals IC50 divided by one plus the labeled ligand concentration over its Kd. Ki is a property of the compound and receptor and can be compared across experiments. Understanding curve fitting, including the Hill slope, is covered in our dose-response guide.
Traps for peptide researchers
- Adsorption: peptides stick to tubes and filters, so low-binding plastics and carrier protein help. See our adsorption post.
- Degradation: membranes contain proteases that can chew the peptide, so protease inhibitors and short incubations are typical.
- Depletion: if too much ligand binds, the free concentration is no longer what you think.
- Equilibrium: slow binders need longer incubations, and checking time courses is wise.
- Filter binding: pre-soaking filters can reduce background.
Binding is not function
A compound can bind tightly and do nothing, or act as a blocker. To learn whether a compound activates a receptor, researchers run a functional assay as well, such as a cAMP assay. Some ligands also activate one signaling path more than another, which is the subject of biased agonism. For a family example, see the melanocortin receptors.
The bottom line
Radioligand binding is simple in concept and demanding in practice. Good controls, careful math and honesty about limits give numbers that can be trusted. Explore the receptor biology in our Research Library.
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For laboratory research use only. Not for human or veterinary use, and not intended to diagnose, treat, cure or prevent any condition. This article is educational and is not medical advice.