A binding experiment returns numbers whether or not the underlying interaction is real. This page explains what the common parameters mean, which artifacts produce convincing but misleading data, and what a careful reader or reviewer will expect to see reported.
| Parameter | Units | What it describes |
|---|---|---|
| KD, equilibrium dissociation constant | Molar (M) | The concentration at which half the binding sites are occupied at equilibrium. Smaller means tighter. |
| ka or kon, association rate | M−1s−1 | How quickly the complex forms once the partners meet. |
| kd or koff, dissociation rate | s−1 | How quickly the complex falls apart. |
| Residence time | Seconds | 1 / kd. The average lifetime of the complex once formed. |
| IC50 | Molar (M) | Competitor concentration that halves the signal. Depends on the assay conditions, so it is not an intrinsic constant. |
| Stoichiometry (n) | Dimensionless | Binding sites per molecule. Available from ITC and a valuable sanity check. |
KD is related to the rates by KD = kd / ka. Two peptides can share a KD and behave very differently: one binding and releasing quickly, the other binding slowly and staying bound. For inhibitor work the residence time often predicts cellular behaviour better than affinity alone, which is why kinetic methods are worth the extra effort when the goal is a functional molecule.
Every affinity number inherits the accuracy of the concentrations used to calculate it. This is the most common invisible error in peptide work, and it is worth stating plainly: a KD is only as good as the peptide concentration behind it.
Lyophilized peptide is not pure peptide by weight. It contains bound water and counter-ions, and the fraction that is actually peptide — the net peptide content — varies with sequence and purification history. Dissolving "1 mg" of powder in 1 mL does not give a 1 mg/mL peptide solution. If the true content is 70 %, every affinity constant derived from that stock is out by the same factor.
For quantitative work, determine concentration by absorbance at 280 nm if the peptide contains tryptophan or tyrosine, or by amino acid analysis if it does not. See calculating peptide concentration and net peptide content.
If either partner is multivalent — an antibody with two arms, a dimeric protein, or a densely coated surface presenting many copies of the peptide — the measured affinity will be tighter than the true single-site value, and dissociation will look unrealistically slow. The diagnostic is surface density: prepare several densities and check whether the fitted KD changes. If it does, you are measuring avidity.
Aggregating peptides produce steep, cooperative-looking curves, apparent stoichiometries that make no structural sense, and poor reproducibility between preparations. A Hill slope substantially above one in a system with no reason for cooperativity is a warning sign. So is a result that shifts when the stock is diluted differently or after a freeze-thaw cycle. Dynamic light scattering or a simple centrifugation step before the measurement will often settle the question.
Highly cationic peptides, including many cell-penetrating sequences, bind negatively charged surfaces, dextran matrices and plasticware. The resulting signal can be dose-dependent and perfectly reproducible without reflecting a specific interaction. Raising the salt concentration moderately, using a reference surface and testing a scrambled peptide of identical charge all help to distinguish the two.
In surface methods, a sample buffer different from the running buffer produces a square-shaped jump at injection and again at the end. In calorimetry, mismatched buffers produce heats that have nothing to do with binding. DMSO is the usual culprit in both cases: it must be matched closely across all samples, including the controls.
When the target concentration is comparable to or above the KD, free ligand is measurably depleted by binding, and the simple fitting equation no longer applies. The fitted KD then collapses towards the target concentration regardless of the true affinity. Working below the expected KD, or using a fitting model that accounts for depletion, avoids reporting a number that is really just your target concentration.
Weak binding is a result
A peptide that binds at high micromolar affinity has not failed. Isolated linear motifs frequently bind in that range, and such interactions are biologically important when they occur at high local concentration or in multivalent arrays. The honest course is to report the number with its uncertainty and the method used, rather than to search for conditions that produce a more attractive one.
Reviewers increasingly expect the detail that makes a measurement reproducible. A complete report usually includes:
Keeping the certificate of analysis for each peptide batch alongside the data makes this straightforward to assemble later, and answers most questions a reviewer is likely to raise about the material itself.
Paste a peptide or protein sequence to review length, charge, hydrophobicity and other properties before you order.
Research use only. LifeTein peptides and proteins are supplied for laboratory research use. They are not for human or veterinary use, and not for diagnostic or therapeutic use. See our research use policy.
If you need peptides with accurately determined content for quantitative binding work, or a binding measurement performed for you, email sales@lifetein.com or use our quotation form.