pI Calculator
(v1.0.0)Docs
Calculate the theoretical isoelectric point (pI) - the pH at which a protein has no net charge.
Paste a protein sequence or several FASTA records to calculate the theoretical isoelectric point (pI) in the browser. The results table gives one row per sequence, with its identifier, length and pI. This calculator uses a fixed Henderson-Hasselbalch model with EMBOSS pKa values and free N and C termini.
For example, the human insulin B chain is:
FVNQHLCGSHLVEALYLVCGERGFFYTPKTIt returns Sequence, 30 amino acids and pI 7.41. This is the isolated, unmodified chain with reduced cysteines, not the disulfide-linked insulin molecule.
Select Human insulin chains to load both chains, or paste:
>insulin-B human insulin B chain
FVNQHLCGSHLVEALYLVCGERGFFYTPKT
>insulin-A human insulin A chain
GIVEQCCTSICSLYQLENYCN| Protein ID | Number of amino acids | Isoelectric point |
|---|---|---|
| insulin-B | 30 | 7.41 |
| insulin-A | 21 | 3.61 |
| Input | Requirements |
|---|---|
| Plain sequence | One sequence, optionally wrapped across lines. Spaces and lowercase letters are accepted. |
| FASTA | Each >header must be followed by sequence data on the next line. Descriptions stay in the header. Each record gets its own result. |
| CSV or TSV file | A header row with id and sequence columns. Quoted cells and extra columns are supported. |
| PDB file | Observed protein residues from the first model, with one result per chain. A warning explains that unresolved residues are omitted and each chain receives free termini. |
| Uploads | .txt, .csv, .fasta, .pdb, .fa, .fas, up to 50 MiB per file. |
Use the 20 standard one-letter amino acid codes: ACDEFGHIKLMNPQRSTVWY. Invisible characters introduced by document copying are normalized. Empty records, position numbers, stop markers, alignment gaps, and ambiguous or nonstandard residues are rejected rather than silently removed. Resolve these before calculating pI. Names that contain only amino acid letters cannot be distinguished from sequence text; put names in FASTA headers.
There are no calculation settings. The pKa values, free-terminal assumptions and numerical tolerance below are fixed.
| Column | Meaning |
|---|---|
proteinId | First token of the FASTA header, or the accession from a UniProt sp|accession|name or tr|accession|name header. Plain input uses Sequence. |
numAminoAcids | Number of residues in that sequence or observed PDB chain. |
isoelectricPoint | Theoretical zero-net-charge pH, reported to two decimal places. |
Each input record receives its own result. The interactive table sorts by protein ID initially and supports column sorting. Use its download action to export the rows. An invalid sequence stops the calculation so a missing result cannot be mistaken for a complete batch.
The isoelectric point (pI) is the pH at which a protein carries no net electrical charge. At this pH, the positive and negative charges on ionizable amino acid side chains and termini are perfectly balanced, resulting in a neutral molecule.
Near the pI, reduced electrostatic repulsion can promote aggregation and lower solubility. Solubility also depends on the protein, buffer and temperature. Isoelectric focusing uses a pH gradient to concentrate proteins near their pI values.
For practical applications, the pI guides buffer selection for protein purification, predicts migration in electrophoresis, and informs formulation strategies for biopharmaceuticals.
For pI alongside molecular weight and extinction coefficient, use Protein Parameters. To turn an A280 reading into protein concentration, use the extinction coefficient calculator.
The calculation uses the Henderson-Hasselbalch equation to determine the net charge of a protein at any given pH. For each ionizable group, the fractional charge depends on the pH and the group's dissociation constant (pKa).
Q = 1 / (1 + 10^(pH - pKa)).Q = -1 / (1 + 10^(pKa - pH)).The total net charge is the sum of contributions from all ionizable groups in the protein. The isoelectric point is the pH where this sum equals zero.
Seven amino acids contribute charged side chains that determine protein pI:
Additionally, the N-terminus (amino group) and C-terminus (carboxyl group) contribute positive and negative charges, respectively. For short peptides, these terminal charges can dominate the overall pI.
The fixed pKa values match the table in the EMBOSS iep documentation. The N-terminal value is 8.6 for every sequence. This is not the Bjellqvist model, which uses different constants and terminal-residue corrections. EMBOSS installations can use other pKa data files, so compare the constants when comparing results.
| Group | pKa | Group | pKa |
|---|---|---|---|
| N-terminus | 8.6 | C-terminus | 3.6 |
| Lysine (K) | 10.8 | Aspartate (D) | 3.9 |
| Arginine (R) | 12.5 | Glutamate (E) | 4.1 |
| Histidine (H) | 6.5 | Cysteine (C) | 8.5 |
| Tyrosine (Y) | 10.1 |
The search starts between pH 0 and 14. At each midpoint, a positive net charge moves the lower bound up; a negative charge moves the upper bound down. The interval is halved until it is at most 0.01 pH unit wide. The midpoint is then rounded to two decimals. If zero net charge lies outside the search range, the calculation reports an error instead of a boundary value. Numerical convergence and display precision do not establish experimental accuracy.
A sequence rich in Asp and Glu generally has a lower pI, while Lys and Arg tend to raise it. Below the predicted pI the model gives positive net charge; above it the model gives negative net charge. The estimate describes the exact sequence entered, so signal peptides, affinity tags and missing residues can change it.
Understanding protein pI is essential for several experimental workflows.
Chromatography buffer design: Below its pI a protein generally has positive net charge and can bind a cation exchanger. Above its pI it generally has negative net charge and can bind an anion exchanger. Binding also depends on salt, resin and surface charge distribution.
Isoelectric focusing (IEF): A pH gradient drives proteins toward their zero-charge positions. The calculated pI can help select an initial gradient range.
Solubility and formulation: Testing buffers away from the estimated pI can help assess aggregation, but sequence pI alone does not determine a suitable formulation.
Electrophoresis interpretation: Native electrophoresis depends on charge, size and shape. The pI helps interpret the expected charge sign at the gel's pH.
The calculated pI is theoretical and assumes ideal conditions. Several factors can cause discrepancies with experimental values:
Modifications: Phosphorylation, acetylation and charged glycans can shift the observed pI. The calculator does not model modifications or blocked termini.
Disulfide bonds: All cysteines are treated as reduced thiols whose fractional negative charge depends on pH. Cysteines in disulfide bonds do not contribute free thiols. No disulfide correction is applied.
Environment and structure: Fixed pKa values ignore local electrostatic interactions, solvent accessibility, folding, salt and temperature. PDB input supplies a sequence, not a structure-dependent charge calculation.
| Goal | Tool |
|---|---|
| Quick fixed-model estimate with one row per sequence | This pI Calculator |
| Compare pKa scales or use trained protein/peptide models | IPC 2.0 |
| Broader sequence property report | Protein Parameters |
ExPASy uses the Bjellqvist model. This calculator uses the fixed constants above, including an N-terminal pKa of 8.6. Different pKa sets and terminal assumptions can produce different theoretical values for the same sequence.
Yes, by uploading a .pdb file. The result describes each observed protein chain from the first model. For the full-length protein, use its complete FASTA sequence because unresolved residues are missing from coordinate-derived sequences.
Its predicted net charge is zero. Positive and negative groups can still be charged, and local surface charge can remain uneven.
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Calculate mean Kyte-Doolittle hydropathy and compare protein sequences.
Calculate dipeptide-based instability scores and predicted stability for protein sequences.
Calculate the molecular weight of a protein or peptide sequence in Da and kDa.
Calculate the percentage and frequency of each amino acid in protein sequences
Scan protein sequences for glycosylation, phosphorylation, localization signals, and other biologically important motifs.
Analyze aggregation-prone regions in a protein structure.
Plot net charge vs pH to visualize protein charge behavior and identify the isoelectric point.