What does this calculator estimate?

It estimates a single-point Kd for protein–DNA binding under a 1:1 equilibrium model.

Can I enter a bound fraction directly?

Yes. Select Fraction bound and enter a value strictly between 0 and 1.

How does anisotropy mode calculate the bound fraction?

It uses f = (r − r free) / (r bound − r free), based on the measured, free-state, and bound-state anisotropy values.

What does ΔG° represent here?

ΔG° is the binding free-energy estimate calculated from Kd at the selected temperature.

Is this a replacement for fitting a full binding curve?

No. It is a single-point estimate; a complete titration should be fitted as a full curve for more robust analysis.

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Protein–DNA Binding Affinity Calculator (Kd Estimate)

Single-point 1:1 Kd estimate: Kd = [P] × (1−f)/f from a direct fraction or anisotropy-derived fraction, plus ΔG° = RT·ln(Kd) and affinity bands from very tight (<1 nM) to weak (>1 µM). Cantor & Schimmel / Heyduk & Lee / Lakowicz derived. Educational use only.

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2 examples

f = 0.5 at 50 nM protein → Kd = 50 nM, ΔG° = −9.96 kcal/mol

An EMSA or anisotropy point at 50 nM protein shows half the labeled DNA bound. Since f = 0.5, Kd = [P] = 50 nM directly — a tight, sequence-specific interaction. At 25 °C this equals ΔG° = RT·ln(5×10⁻⁸ M) ≈ −9.96 kcal/mol, comfortably in the range of good transcription-factor target sites.

{"result":"Kd ≈ 50 nM (fraction bound 0.5 at 50 nM protein); ΔG° = −9.96 kcal/mol at 25 °C.","metadata":{"input":{"mode":"fraction","proteinConcNm":50,"fractionBound":0.5,"temperatureC":25},"result":{"kdNm":50,"fractionBound":0.5,"deltaGKcalMol":-9.96}}}

Anisotropy 0.18 between 0.12/0.25 → f = 0.46, Kd = 23.33 nM

A fluorescein-labeled oligo gives r_free = 0.12 alone and r_bound = 0.25 saturated with protein. A sample at 20 nM protein reads r = 0.18, so f = (0.18−0.12)/(0.25−0.12) = 0.46 and Kd = 20 × 0.54/0.46 = 23.33 nM (ΔG° = −10.41 kcal/mol at 25 °C). The example shows the anisotropy-mode algebra and the practical tip that r_free and r_bound must be measured in the same buffer.

{"result":"Kd ≈ 23.33 nM (fraction bound 0.46 from anisotropy 0.18); ΔG° = −10.41 kcal/mol.","metadata":{"input":{"mode":"anisotropy","proteinConcNm":20,"anisotropy":0.18,"anisotropyFree":0.12,"anisotropyBound":0.25},"result":{"kdNm":23.33,"fractionBound":0.46,"deltaGKcalMol":-10.41}}}

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8 options

SettingsAdjust formats, ranges, numbers, and modes.8

Calculation ModeselectRequiredEnter the bound fraction directly, or derive it from anisotropy values.Protein Concentration (nM)numberRequiredTotal protein concentration in the binding reaction (kept well above the labeled DNA).Fraction Bound (0–1)numberRequiredFraction of labeled DNA in complex (fraction mode; strictly between 0 and 1).Measured Anisotropy (r)numberRequiredAnisotropy of the sample (anisotropy mode).Free-State Anisotropy (r free)numberRequiredAnisotropy of the unbound labeled DNA alone.Bound-State Anisotropy (r bound)numberRequiredAnisotropy of the fully bound complex (protein-saturated).Temperature (°C)numberRequiredIncubation temperature, used for ΔG° = RT·ln(Kd).Decimal PlacesnumberOptional

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Protein–DNA Binding Affinity Calculator (Kd Estimate)

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1. Half-bound DNA at 50 nM protein

2. Anisotropy-based Kd estimate

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