Protein Isoelectric Point Calculator

Formula used

This calculator uses a charge-balance model built from Henderson–Hasselbalch relations for ionizable groups. The net charge is the sum of N-terminus, C-terminus, and side-chain contributions.

The isoelectric point is the pH where the computed net charge crosses zero. A bisection search is used over pH 0–14 to find the pH that minimizes |net charge|.

How to use this calculator

1. Paste the protein sequence using one-letter amino-acid codes.
2. Select a pKa preset, or enable custom overrides if needed.
3. Choose a pH range and step size for the charge table.
4. Click Submit to compute pI, net charge, and the table.
5. Use Download CSV or Download PDF to export results.

Professional article

1) Isoelectric point and biophysical meaning

The isoelectric point (pI) is the pH at which a protein’s net electrical charge is zero. At this condition, electrostatic repulsion is reduced, solubility can drop, and aggregation risk may increase. In electrophoresis and isoelectric focusing, proteins migrate until they reach their pI, enabling separation based on charge characteristics.

2) Ionizable groups included in the model

This calculator models common ionizable sites: N-terminus, C-terminus, and side chains of Asp (D), Glu (E), Cys (C), Tyr (Y), His (H), Lys (K), and Arg (R). The sequence is parsed into residue counts, then each ionizable site contributes a pH-dependent fractional charge. This is a practical approximation for many workflows.

3) Henderson–Hasselbalch charge fractions

For basic groups, the protonated fraction follows 1/(1+10^(pH−pKa)), contributing positive charge. For acidic groups, the deprotonated fraction follows 1/(1+10^(pKa−pH)), contributing negative charge. Summing these terms yields the net charge curve across the chosen pH range, with steep transitions near each pKa.

4) pKa set selection and sensitivity

Different pKa compilations reflect experimental conditions and reference choices. Small pKa shifts can move pI noticeably, especially for proteins rich in ionizable residues. The preset selector allows side-by-side comparison, while custom overrides support laboratory-specific assumptions, such as altered terminal pKa in structured proteins or modified residues.

5) Numerical search used to find pI

The pI is computed by searching pH 0–14 for the zero-charge point. A bisection method repeatedly narrows the interval where the sign of net charge changes, providing stable convergence. If a strict sign change is not observed, the routine still reports the pH that minimizes the absolute net charge within the interval.

6) Interpreting the charge table output

The generated pH table reports net charge at each step, which is useful for buffer planning. For example, operating one pH unit away from pI often increases net charge magnitude, improving solubility and reducing precipitation. Tracking the sign change helps confirm whether the chosen pH bracket contains the pI and whether the curve is monotonic in that region. A finer step size can reveal multiple inflection regions driven by clustered pKa values.

7) Practical experimental context

In ion-exchange chromatography, predicted charge at a working pH can guide resin choice and elution strategy. In protein formulation, avoiding pH values near pI can reduce aggregation for some proteins. In isoelectric focusing gels, the pI estimate helps select appropriate gradient ranges for clearer band separation.

8) Limitations and quality checks

This approach assumes independent ionization and does not explicitly model microenvironments, salt effects, post-translational modifications, or strongly coupled residues. For critical decisions, compare multiple pKa sets, validate against experimental pI when available, and review whether the sequence contains unusual residues or modifications that change ionization behavior.