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DNA, RNA & Protein

Protein Isoelectric-Point Model Workbench

Estimate the pH of zero modeled net charge for one unmodified protein sequence and inspect every ionizable-group contribution at pI and a visitor-selected pH.

Biology · experimental measurements

Make the pKa set, terminal corrections and charge-balance root visible instead of presenting pI as an unexplained sequence score.

Private calculations in your browser · explicit inputs and model boundaries
Example preview · Acidic sequenceModeled net-charge curve
-90-6.53.5-47-1.510.50.99914pHModeled net charge

The curve crosses zero at pH 3.19126071. It represents independent ionizable groups under the displayed parameter set.

  1. 1EnterProvide the known values
  2. 2CalculateResults update automatically
  3. 3VerifyReview the details and units
Try an example

Use the 20 standard one-letter amino-acid codes for one unmodified chain.

Enter 0–14.

Calculation result

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Enter valid values to see the result.

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Understand the relationship

The reasoning behind the result

pI is a modeled charge-balance root

Σ qᵢ(pH) = 0

Henderson–Hasselbalch fractions are summed across the free termini and ionizable side chains.

Bisection finds the pH where that explicit model crosses zero net charge.

The parameter set matters

This workflow uses Bjellqvist-style pKa values, including first- and last-residue terminal adjustments.

A different parameter set, temperature, solvent or empirical method can produce a different estimate.

Sequence charge is environment dependent

Local structure, neighboring groups, ligand binding and solution conditions can shift microscopic pKa values.

The curve is an ideal independent-group model, not a titration measurement.

Small and highly basic proteins need care

Limited buffer capacity makes small-chain estimates sensitive, and highly basic proteins lie outside much of the experimental range used to develop the model.

Modifications and blocked termini can materially change the balance.

Follow the numbers

Find the modeled pI of an acidic sequence

  1. Enter MDDDDEEEEAA as one unmodified chain with free termini.
  2. Count the N terminus, C terminus and every D, E and other ionizable side chain.
  3. Calculate each fractional charge at a trial pH.
  4. Sum the positive and negative contributions.
  5. Bisect pH 0–14 until the modeled net charge is effectively zero.

The reported pI is the root of the displayed parameter model, not an experimental 2-D gel position.

Quick guide

How to use this calculator

  1. Enter the exact mature unmodified chain that you intend to model.
  2. Set a comparison pH relevant to your separate experimental context.
  3. Review the charge curve and group ledger before using the pI estimate.

Calculation method

Calculation and interpretation

Make the pKa set, terminal corrections and charge-balance root visible instead of presenting pI as an unexplained sequence score.

For each basic group q=+n/(1+10^(pH−pKa)); for each acidic group q=−n/(1+10^(pKa−pH)). The estimated pI is the pH where the summed modeled charge is zero.

Worked example

Find the modeled pI of an acidic sequence

The reported pI is the root of the displayed parameter model, not an experimental 2-D gel position.

For each basic group q=+n/(1+10^(pH−pKa)); for each acidic group q=−n/(1+10^(pKa−pH)). The estimated pI is the pH where the summed modeled charge is zero.

Supported inputs

Precision and limits

Model estimate

The pI is calculated from a named pKa parameter set and independent-group assumptions, not measured.

Free unmodified chain

Blocked termini, modifications, disulfides, ligands, prosthetic groups and cleavage outside the entered sequence are excluded.

Environment omitted

Temperature, ionic strength, solvent, conformation and neighboring-group pKa shifts are not modeled.

Known difficult ranges

Very small and highly basic proteins can have less reliable model estimates.

No separation guarantee

The result does not predict solubility, focusing behavior, migration, aggregation or purification success.

Numerical and pH support

Sequences are limited to 100,000 standard residues. The root search is limited to pH 0–14 and stops explicitly when modeled charge does not cross zero in that interval.

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