Protein isoelectric point (pI) calculator
Paste a protein sequence in one-letter code. The calculator counts the ionisable groups — the two ends and the side chains of Lys, Arg, His, Asp, Glu, Cys and Tyr — and finds the pH at which their charges cancel out. It also gives the net charge at the pH you enter, 7 by default.
Runs in your browser: nothing you enter is sent anywhere.
How to use it
- Paste the protein or peptide sequence in one-letter code.
- Optionally change the pH for the net charge.
- Read the pI, the net charge and the counts of charged residues.
How it works
Net charge at a given pH:
Z = Σ (basic) 1 ÷ (1 + 10^(pH − pKa)) − Σ (acidic) 1 ÷ (1 + 10^(pKa − pH))
- Basic: the N-terminus, Lys, Arg, His
- Acidic: the C-terminus, Asp, Glu, Cys, Tyr
The pI is the pH where Z = 0, found by bisection between pH 0 and 14 to well below 0.01 of a unit.
Worked example
Glycine alone: (8.6 + 3.6) ÷ 2 = 6.1. Lysine adds a basic side chain, which lifts the pI to 9.7, and aspartate an acidic one, which lowers it to 3.75.
The default sequence, the B chain of human insulin, has 1 Lys, 1 Arg and 2 His against 2 Glu, 2 Cys and 2 Tyr: pI 7.42, net charge +0.4 at pH 7.
Tips
- Choosing a buffer. For ion-exchange chromatography, a buffer about one unit above the pI makes the protein negative (anion exchanger), one unit below makes it positive (cation exchanger).
- Solubility. Proteins tend to aggregate near their pI; keep stock solutions away from it.
- Free cysteines only. Cysteines in disulfide bonds do not ionise; the calculator counts every Cys, so a protein full of disulfides may have a slightly higher real pI.
Frequently asked questions
What is the isoelectric point?
The pH at which a molecule carries no net electric charge. Below its pI a protein is positively charged, above it negatively charged; at the pI it does not move in an electric field and is often least soluble.
How is the pI calculated?
Each ionisable group is given a pKa. By the Henderson–Hasselbalch equation, a basic group carries +1 ÷ (1 + 10^(pH − pKa)) and an acidic group −1 ÷ (1 + 10^(pKa − pH)). The net charge falls steadily as pH rises, and the calculator searches for the pH where it is exactly zero.
Which pKa values are used?
Those of the EMBOSS iep program: N-terminus 8.6, C-terminus 3.6, Lys 10.8, Arg 12.5, His 6.5, Asp 3.9, Glu 4.1, Cys 8.5 and Tyr 10.1. Other tools use other sets (ExPASy uses Bjellqvist values that depend on position), so results can differ by a few tenths of a pH unit.
How accurate is a theoretical pI?
It is an estimate. In a folded protein neighbouring groups shift each other’s pKa, and modifications such as phosphorylation add charges. Treat it as a guide and measure it (by isoelectric focusing) when the exact value matters.
Why does the pI of glycine come out as 6.1?
A single glycine has only its two ends. Their charges cancel exactly halfway between the two pKa values: (8.6 + 3.6) ÷ 2 = 6.1. Textbooks quote 5.97 for glycine because they use its own measured pKa values, 2.34 and 9.60.