What pKa values does this tool use?

The tool uses the widely accepted EMBOSS/ExPASy pKa set: N-terminus 8.0, C-terminus 3.1, Asp (D) 3.9, Glu (E) 4.1, Cys (C) 8.3, Tyr (Y) 10.1, His (H) 6.5, Lys (K) 10.5, Arg (R) 12.5. These match the values used by ExPASy ProtParam and EMBOSS Pepstats.

How is net charge calculated at a given pH?

For each ionizable group, the Henderson-Hasselbalch equation gives the fraction in each protonation state. Basic groups (N-term, His, Lys, Arg) contribute positive charge when protonated; acidic groups (C-term, Asp, Glu, Cys, Tyr) contribute negative charge when deprotonated. The sum of all partial charges is the net charge.

Why might my result differ from ExPASy ProtParam?

Small differences (±0.1 pH units) can arise from different pKa sets or bisection precision. The Lehninger pKa set assigns slightly different values; toggle between EMBOSS and Lehninger in the tool to compare.

Does the tool account for disulfide bonds?

No. Disulfide-bonded cysteines lose their ionizable thiol group. If your peptide contains disulfide bonds, remove the paired Cys residues from your charge calculation or treat their pKa as very high (effectively non-ionizable).

What is the isoelectric point used for in practice?

The pI is used to select running conditions for isoelectric focusing gels, optimize buffer pH for crystallization, predict solubility (minimum near pI), and design ion-exchange chromatography steps where the protein must be either positively or negatively charged.

Isoelectric Point Calculator

Name: Isoelectric Point Calculator
Availability: InStock
Author: Nham Vu

Enter a peptide sequence in single-letter codes to calculate its isoelectric point (pI) and net charge at any pH.

Peptide / Protein Sequence

Enter single-letter amino acid codes (e.g. ACDEFGHIKLM). Spaces and line breaks are ignored.

pKa set: EMBOSS / ExPASy Lehninger Bjellqvist

Isoelectric Point (pI) pH at zero net charge

Net Charge at pH 7.4 physiological pH

Ionizable Groups including termini

Net Charge at a Specific pH

pH:

Net charge:

Charge vs. pH Curve

Ionizable Group Breakdown

Group	Type	Count	pKa	Charge at pI	Charge at pH 7.4

Enter a sequence above and click Calculate pI to see the isoelectric point.

Summary

Enter a peptide sequence in single-letter codes to calculate its isoelectric point (pI) and net charge at any pH.

How it works

Enter your peptide sequence using IUPAC single-letter amino acid codes (e.g., ACDEFGHIKLMNPQRSTVWY).
The tool identifies all ionizable groups: the free N-terminus (pKa 8.0), the free C-terminus (pKa 3.1), and the side chains of D, E, C, Y, H, K, and R.
For each pH from 0 to 14, the Henderson-Hasselbalch equation is applied to each ionizable group to compute its partial charge.
All partial charges are summed to give the net charge at that pH.
The isoelectric point (pI) is the pH where the net charge crosses zero, found by bisection search to four decimal places.
An interactive charge-vs-pH curve is rendered so you can see how charge changes across the full pH spectrum.

Use cases

Predict the optimal pH for protein precipitation or isoelectric focusing (IEF).
Select appropriate buffer conditions for chromatography or electrophoresis.
Estimate solubility — proteins are least soluble near their pI.
Design peptides with a desired charge state at physiological pH.
Verify pI values for recombinant proteins before purification.
Support biochemistry coursework and lab planning.
Quickly compare pI values of protein mutants differing in charged residues.

Frequently Asked Questions

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Last updated: 2026-05-29 · Reviewed by Nham Vu