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A receptor binding assay is how the affinity described in agonist and antagonist terminology actually gets measured in the lab. Saying a compound “binds tightly” is a qualitative claim until it is backed by a number, and receptor binding assays are the standard method for generating that number. This guide explains what a binding assay actually measures and how the resulting values, Kd and IC50, are calculated and interpreted.
What Is a Receptor Binding Assay? How Researchers Measure Affinity
Calling a compound an agonist or an antagonist describes what it does at a receptor. It does not say how strongly it binds. That number, affinity, comes from a specific experimental method: the receptor binding assay. This is how “binds tightly” becomes an actual, reportable value.
What a Binding Assay Measures
A receptor binding assay directly measures how a ligand interacts with its receptor, typically using a radiolabeled version of a known ligand (a radioligand) as a tracer. By measuring how much of that radioligand binds to receptor preparations under different conditions, researchers can calculate the equilibrium dissociation constant, or Kd, a direct measure of how tightly the radioligand binds [1].
Research framing: This article explains a laboratory analytical method in an educational, research context. Compounds referenced elsewhere on this site are supplied by Badger Compounds for laboratory research use only and are not intended for human or veterinary use. Nothing here is medical advice.
Two Types of Binding Experiment
Saturation Binding
Increasing concentrations of a radioligand are tested against a fixed amount of receptor, directly determining Kd and the total number of binding sites (Bmax) [1].
Competition Binding
A fixed concentration of radioligand is tested alongside increasing concentrations of an unlabeled test compound, measuring how well that compound displaces the tracer [1].
Competition binding is the more common approach for characterizing a new compound, since it does not require radiolabeling every candidate, only the single reference tracer.
From IC50 to Ki: The Cheng-Prusoff Equation
A competition binding experiment directly produces an IC50, the concentration of test compound needed to displace half of the bound radioligand. IC50 is useful but assay-dependent, since it varies with the specific radioligand concentration used. To convert IC50 into Ki, a more fundamental value describing the compound’s true affinity for the unoccupied receptor, researchers apply the Cheng-Prusoff equation, published in 1973 and still the standard method for this conversion [2].
IC50 tells you about one specific experiment. Ki, calculated via the Cheng-Prusoff equation, tells you about the compound’s underlying affinity, in a form that can be compared meaningfully across different labs and assay conditions.
Affinity Terms at a Glance
| Term | What it represents |
|---|---|
| Kd | Equilibrium dissociation constant of the radioligand itself, from saturation binding |
| Bmax | Total number of available binding sites, from saturation binding |
| IC50 | Concentration of test compound that displaces 50% of bound radioligand |
| Ki | True affinity constant of the test compound, calculated from IC50 via Cheng-Prusoff |
Why This Connects to Agonist vs Antagonist
A binding assay on its own only measures affinity, not efficacy. It tells you how tightly a compound occupies a receptor, not whether that occupation activates the receptor or simply blocks it. Determining agonist versus antagonist behavior requires a separate functional assay measuring the downstream cellular response, used alongside binding data rather than in place of it. For the underlying framework this builds on, see our post on agonist vs antagonist.
Research Concepts Related to Receptor Binding Assays
Radioligand binding Saturation binding Competition binding Kd and Bmax Cheng-Prusoff equation Binding affinity vs functional efficacyExplore Research-Grade Peptides – ≥99% (HPLC)
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Browse the Catalog- Hulme EC, Trevethick MA. Ligand binding assays at equilibrium: validation and interpretation. Br J Pharmacol. 2010;161(6):1219-1237. PMID 20132208
- Cheng Y, Prusoff WH. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol. 1973;22(23):3099-3108. PMID 4202581
Disclaimer: This article is for informational and educational purposes only. Products discussed elsewhere on this site are research use only, not for human consumption, veterinary use, clinical use, or any consumer application. Statements have not been evaluated by the FDA. This content does not provide medical advice.
