Protein Isoelectric Point (pI) Calculator
Estimate a protein’s isoelectric point from the counts of its ionizable amino acids using Henderson–Hasselbalch.
How it works
Net charge(pH) = Σ positive/(1+10^(pH−pKa)) − Σ negative/(1+10^(pKa−pH)); pI is the pH where net charge = 0
Each ionizable group carries a pH-dependent charge set by its pKa through the Henderson–Hasselbalch relation: basic groups (N-terminus, His, Lys, Arg) are protonated and positive at low pH, while acidic groups (C-terminus, Asp, Glu, Cys, Tyr) lose protons and turn negative at high pH. Adding all these fractional charges gives a net-charge curve that falls smoothly with pH, and the isoelectric point is simply the pH where it crosses zero. A protein is least soluble and does not move in an electric field at its pI, which is why it matters for purification and 2-D gels.
Worked example
A protein with 5 Asp, 5 Glu, 1 Cys, 3 Tyr, 2 His, 6 Lys and 4 Arg (plus its two termini) has its positive and negative contributions balancing near pH 8.3 — that pH is its estimated pI, and at pH 7 it carries a small net positive charge.
Sources: Isoelectric point — Wikipedia
Frequently asked questions
Why does the answer use only some amino acids?
Only side chains that gain or lose a proton over the relevant pH range affect charge. The other 13 amino acids are electrically neutral, so they do not shift the isoelectric point.
How accurate is this estimate?
It uses average pKa values and ignores local structure, which can shift a group’s pKa by a unit or more. Expect it within a few tenths of a pH unit of a measured pI for most soluble proteins.
What happens to the protein at its pI?
Its net charge is zero, so it stops migrating in an electric field and its solubility is at a minimum — the basis of isoelectric focusing and of pI-based precipitation.
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This calculator is for educational purposes. Double-check important results before acting on them.