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Isoelectric Point Calculator

Chemistry
One-letter (YGGFL, ProForma S[Phospho]), three-letter (H-Tyr-Gly-Gly-Phe-Leu-OH) or FASTA. Spaces, digits and line breaks are ignored.

Try:

read as one-letter

N-terminus

C-terminus

Ring

Fixed modifications

Disulfide bonds

Number of S–S bonds

0 to 0 (free Cys ÷ 2). ExPASy and most calculators count every Cys as free.

pKa scale

Bjellqvist et al. 1994, Electrophoresis 15:529, as used by ExPASy Compute pI/Mw, Biopython and pyteomics. Calibrated on immobilised pH gradient gels, roughly pH 4-7.

pH for “charge at pH”

Isoelectric point · Bjellqvist

pI 6.56

Net charge at pH 7.00

−0.40

Across 5 scales

6.56–7.68

Net charge vs pH

Left of the pI: net +
Right of the pI: net −
Near-neutral window (|charge| ≤ 0.5)

Click the chart to set the pH.

Ion exchange and solubility hint

Cation exchange: run at pH ≤ 5.56 (pI − 1), where the net charge is +1.28. Anion exchange: pH ≥ 7.56 (pI + 1), where it is −0.78. Solubility is usually lowest near the pI.

At pH 7.00: net negative → moves toward the anode in electrophoresis and binds anion exchangers.

A rule of thumb, not a prediction: some protocols use ±0.5, and real proteins bind through surface patches. Make a buffer at your pH →

Groups at pH 7.00

GroupKindCountpKaEachTotal
N-term (Met)base17.00+0.500+0.500
Kbase710.00+0.999+6.993
Rbase412.00+1.000+4.000
Hbase15.98+0.087+0.087
Dacid54.05−0.999−4.994
Eacid64.45−0.997−5.983
Yacid110.00−0.001−0.001
C-term (Gly)acid13.55−1.000−1.000
Net charge−0.398

Scale comparison

56789pHspread 1.11 pH unitsBjellqvist6.56Bjellqvist: pI 6.56EMBOSS7.54EMBOSS: pI 7.54Lehninger7.68Lehninger: pI 7.68Sillero7.31Sillero: pI 7.31Dawson7.13Dawson: pI 7.13

Scales disagree by 1.11 pH units for this sequence. Its charge curve is shallow near the pI, so small pKa differences move the pI a lot.

ScalepICharge at pH 7.00Source
Bjellqvist6.56−0.40Bjellqvist et al. 1994, Electrophoresis 15:529, as used by ExPASy Compute pI/Mw, Biopython and pyteomics. Calibrated on immobilised pH gradient gels, roughly pH 4-7.
EMBOSS7.54+0.23EMBOSS Epk.dat, as listed in the Python peptides package.
Lehninger7.68+0.10Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed. Free-amino-acid values; the termini are alanine's.
Sillero7.31+0.16Sillero & Ribeiro 1989, Anal. Biochem. 179:319.
Dawson7.13+0.04Dawson et al., Data for Biochemical Research, 1989 (termini from Sillero).

Residue charge map at pH 7.00

H₃N⁺ +0.50
1
11
21
31
41
51
61
71
COO⁻ −1.00
Positive (+)
Negative (−)
Not ionizable

Darker means more charge at this pH.

Tiles sum to −0.40 at pH 7.00

Hover or focus a residue tile to read its pKa and charge.

About This Tool

Isoelectric Point Calculator – pI and Net Charge vs pH for Peptides and Proteins

The isoelectric point (pI) is the pH at which a peptide or protein carries no net electric charge. Below it the molecule is positive, above it negative. That single number decides which ion-exchange resin binds your protein, where it runs in isoelectric focusing and why it tends to precipitate when the buffer pH sits close to it, since solubility is usually lowest there.

How the charge is summed

Every group that can gain or lose a proton is treated independently: the N-terminal amine, the C-terminal carboxyl and the side chains of Lys, Arg, His, Asp, Glu, Cys and Tyr. Each contributes a Henderson–Hasselbalch fraction, and the total is

Z(pH) = Σ bases n / (1 + 10^(pH − pKa)) − Σ acids n / (1 + 10^(pKa − pH)) + permanent charge

Because every term falls as pH rises, Z crosses zero exactly once, and a bisection between pH 0 and 14 finds the pI. The curve above the results shows the whole path, with the near-neutral window where the charge stays within ±0.5.

Why the pKa scales disagree

The Bjellqvist scale, used by ExPASy, was calibrated on immobilised pH gradient gels. The Lehninger scale uses pKas measured on free amino acids, and EMBOSS, Sillero and Dawson use other compilations. When a protein has few His residues and a shallow charge curve near neutral pH, small pKa differences move the pI a lot. Ubiquitin is 6.56 on Bjellqvist and 7.68 on Lehninger, a spread of more than one pH unit, so the second decimal is a property of the model, not of the protein.

Termini and modifications

  • Amidation removes the C-terminal negative charge. Amidated substance P has no acidic group at all, so it never reaches neutrality and has no pI.
  • Acetylation of the N-terminus or a lysine removes a positive charge, which lowers the pI.
  • Phosphorylation adds about −2 at pH 7.4, so one phosphoserine can turn a basic peptide acidic.
  • Pyroglutamate formation uses up the N-terminal amine, and for Glu also the side-chain carboxyl.

Disulfide bonds

Free cysteine thiols are weak acids, so pairing them removes a little negative charge. Hen lysozyme comes out at 9.32 when every Cys is counted free, as most calculators do, and at 10.46 when all eight are paired into four disulfides.

Free amino acids

For a single amino acid the pI is the mean of the two pKas on either side of the neutral species. Glycine is (2.34 + 9.60) / 2 = 5.97, and aspartic acid uses its two lower pKas, (1.88 + 3.65) / 2 = 2.77. Lysine and histidine use their two higher ones: 9.74 and 7.59. The peptide scales describe residues inside a chain, so a lone Lys gives a different number on the Peptide tab.

A model, not a measurement
The calculation ignores folding, buried groups and salt bridges. Lysozyme's measured pI is commonly quoted around 11, above every value computed here. Use the result to choose a starting pH, and confirm it experimentally.

Frequently Asked Questions

Is the Isoelectric Point Calculator free?

Yes, Isoelectric Point Calculator is totally free :)

Can I use the Isoelectric Point Calculator offline?

Yes, you can install the webapp as PWA.

Is it safe to use Isoelectric Point Calculator?

Yes, any data related to Isoelectric Point Calculator only stored in your browser (if storage required). You can simply clear browser cache to clear all the stored data. We do not store any data on server.

What is the isoelectric point of a protein or peptide?

The isoelectric point (pI) is the pH at which the molecule carries no net electric charge, because its positive and negative groups cancel. Below the pI the net charge is positive and above it the net charge is negative. Solubility is usually lowest near the pI, and the molecule stops moving in an electric field in isoelectric focusing.

How is the pI calculated from an amino-acid sequence?

The calculator lists every group that can gain or lose a proton: the N-terminal amine, the C-terminal carboxyl and the side chains of Lys, Arg, His, Asp, Glu, Cys and Tyr. At each pH it adds up one Henderson–Hasselbalch fraction per group, then finds the pH where the total charge crosses zero by bisection between pH 0 and 14.

Why do different pKa scales give different pI values, and which should I use?

Each scale assumes different pKa values: Bjellqvist comes from immobilised pH gradient gels, Lehninger from free amino acids, and the others from other compilations. For proteins with few His residues near neutral pH the charge curve is shallow, so a small pKa change moves the pI by up to about 1 unit. Bjellqvist is the default and matches ExPASy; compare the scales to see how model-dependent your value is.

How do phosphorylation, acetylation, amidation and disulfide bonds change the pI?

Phosphorylation adds about −2 charge at pH 7.4, lowering the pI. Acetylating the N-terminus or a Lys removes a positive charge, and amidating the C-terminus removes a negative charge. A disulfide bond removes two Cys thiols, which are weakly acidic, so it raises the pI slightly. The calculator models each of these when you add a modification, a terminus choice or a disulfide count.

Why does the calculated pI differ from the one measured by isoelectric focusing?

The calculation treats every group as independent and fully exposed. A real protein folds, buries some groups, forms salt bridges and binds ions, all of which shift pKa values. Glycosylation, other modifications and the gel conditions add further differences, so a calculated pI is best read as an estimate within about one pH unit.

How do I calculate the pI of a single amino acid?

Use the Free amino acid tab. For an amino acid with a neutral side chain, the pI is the mean of the α-COOH and α-NH₃⁺ pKas. For Lys, Arg and His it is the mean of the two higher pKas, and for Asp and Glu the mean of the two lower ones. For histidine that is (6.00 + 9.17) / 2 = 7.59.