Protein–DNA Binding Affinity Calculator (Kd Estimate)

Protein–DNA Binding Affinity Calculator (Kd Estimate) | Online Free Tool | Elysia Tools

Tool usage guide

Learn when to use this tool, what it supports, and how real users apply it.

Key facts

Category: Education
Input types: select, number
Output type: json
Sample coverage: 4
API ready: Yes

Overview

The Protein–DNA Binding Affinity Calculator estimates a single-point dissociation constant (Kd) for a 1:1 binding model using either a direct bound fraction or fluorescence anisotropy values. It also calculates ΔG° from Kd and groups the result into an affinity band for educational use and experimental planning.

When to use

Estimate Kd from one protein concentration and a measured fraction of labeled DNA bound.
Convert fluorescence anisotropy measurements into a bound fraction and estimate Kd.
Calculate the corresponding ΔG° and review whether the estimated affinity is very tight, strong, moderate, or weak.

How it works

1Choose Fraction bound or Fluorescence anisotropy as the calculation mode.
2Enter the total protein concentration in nM and the temperature in °C.
3For fraction mode, provide a bound fraction between 0 and 1; for anisotropy mode, enter the measured, free-state, and bound-state anisotropy values.
4The calculator applies Kd = [P] × (1−f)/f, calculates ΔG° = RT·ln(Kd), and returns the estimated Kd, bound fraction, free energy, and affinity band.

Use cases

Quickly estimate transcription factor or other protein–DNA binding affinity from an EMSA or related bound-fraction measurement.
Interpret fluorescence anisotropy readings when free and fully bound reference values are available.
Compare estimated Kd and ΔG° values during educational exercises or early experimental planning.

Examples

1. Half-bound DNA at 50 nM protein

Molecular biology student

Background

An experiment indicates that half of the labeled DNA is bound when the protein concentration is 50 nM.

Problem

Estimate the dissociation constant and binding free energy from this single measurement.

How to use

Select Fraction bound, enter 50 nM for protein concentration, 0.5 for fraction bound, and 25 °C for temperature.

{"mode":"fraction","proteinConcNm":50,"fractionBound":0.5,"temperatureC":25,"decimalPlaces":2}

Outcome

The calculator returns Kd ≈ 50 nM and ΔG° ≈ −9.96 kcal/mol at 25 °C.

2. Anisotropy-based Kd estimate

Fluorescence assay researcher

Background

A labeled DNA sample has r free = 0.12, r bound = 0.25, and a measured anisotropy of 0.18 at 20 nM protein.

Problem

Convert the anisotropy reading into a bound fraction and estimate Kd.

How to use

Select Fluorescence anisotropy and enter the measured, free-state, and bound-state anisotropy values along with 20 nM protein and 25 °C.

FAQ

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.

{"mode":"anisotropy","proteinConcNm":20,"anisotropy":0.18,"anisotropyFree":0.12,"anisotropyBound":0.25,"temperatureC":25,"decimalPlaces":2}

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Key facts

Overview

When to use

How it works

Use cases

Examples

1. Half-bound DNA at 50 nM protein

2. Anisotropy-based Kd estimate

FAQ

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