Isoelectric Point Calculator
FREE TOOLAccepts single-letter IUPAC codes. FASTA headers and whitespace are ignored automatically.
⚡ Isoelectric Point Results
Net Charge vs pH (pH 0 – 14)
Charge at a Specific pH
Introduction
This tool predicts the isoelectric point (pI) of a protein directly from its amino acid sequence, using the same Henderson–Hasselbalch-based approach as ExPASy ProtParam. Molecular biologists and protein chemists use it daily to plan isoelectric focusing gels, choose ion-exchange chromatography conditions, and anticipate where a protein will be least soluble during purification.
About the Tool
The Isoelectric Point Calculator is a free, browser-based tool that computes a protein's theoretical pI directly from its amino acid sequence, along with the predicted net charge at pH 7.0, pH 7.4, and any pH you choose, plus a full charge-vs-pH curve across the range 0–14. Every calculation runs client-side in your browser — sequences are never sent to a server or stored, so it is safe to use with unpublished or proprietary sequences.
How to Use This Calculator
The calculator accepts a protein sequence in single-letter amino acid code, either pasted directly or uploaded as a file. Follow these steps to get a result:
>) and whitespace are stripped automatically.Formula Explanation
The isoelectric point is found by summing the predicted charge contribution of every ionizable group in the sequence at a given pH, then solving for the pH where that sum equals zero.
fractional (+) charge = 1 / (1 + 10^(pH − pKa))
Acidic groups (C-terminus, Asp, Glu, Cys, Tyr):
fractional (−) charge = 1 / (1 + 10^(pKa − pH))
Net charge(pH) = Σ(+ contributions) − Σ(− contributions)
The calculator performs a binary search across pH 0–14 to find the pH where the net charge crosses zero — that pH is the pI. All pKa values used are the Lehninger/ExPASy ProtParam scale.
Worked Example
Sample Input
A short peptide containing 2 Lys, 1 Arg, 1 His, 1 Asp, and 1 Glu, plus free N- and C-termini.
Step-by-Step Calculation
This peptide has more basic groups (2 Lys, 1 Arg, 1 His, plus the N-terminus) than acidic groups (1 Asp, 1 Glu, plus the C-terminus), so it carries a net positive charge at low pH. Using the pKa values from the Formula Explanation above, the calculator sums the fractional charge of every group at a series of trial pH values and narrows in on the zero-crossing point using binary search.
Final Result
pI ≈ 9.8
Interpretation
Because the pI is well above physiological pH, this peptide would migrate toward the cathode during isoelectric focusing and would bind well to a cation-exchange resin at pH 7.
Result Interpretation
The pI value tells you the pH at which the protein has no net charge and minimal solubility. The net charge at pH 7.0 and 7.4 values tell you which direction the protein is charged under near-physiological conditions — a positive value means the protein behaves as a cation and will bind cation-exchange resin, while a negative value means it behaves as an anion and will bind anion-exchange resin. The charge-vs-pH curve shows how steeply the charge changes around the pI; a steep curve means small pH changes produce large charge swings, which is useful information when fine-tuning a chromatography gradient. The ionizable residue table lists exactly which groups were counted and their pKa values, so you can see which residues are driving the result.
Practical Applications
Reach for a pI calculator whenever you are planning a method that depends on a protein's charge state. Common lab scenarios include selecting a starting pH for ion-exchange chromatography, choosing focusing ranges for isoelectric focusing (IEF) or 2D-PAGE, predicting whether a recombinant protein will run as expected on a native gel, anticipating solubility problems during concentration or dialysis steps, and comparing the charge profiles of two protein variants (for example, before and after a point mutation) to see how the change shifts the pI.
Scientific Notes & Limitations
This calculator reports a theoretical pI based on isolated side-chain pKa values, not an experimentally measured one. Different tools use different pKa scales — this one uses Lehninger/ExPASy ProtParam values, while others use EMBOSS, Bjellqvist, or Sillero scales — so small differences from values listed on UniProt or ExPASy are expected. The model only accounts for the standard ionizable groups on unmodified amino acids (the N-terminus, C-terminus, and the side chains of Asp, Glu, His, Cys, Tyr, Lys, and Arg); it does not account for disulfide bonds, post-translational modifications such as phosphorylation or glycosylation, bound cofactors, or the protein's actual folded structure and local environment, any of which can shift the true pI by a noticeable amount.
Practical Tips
- Choose a working buffer pH at least 1 pH unit away from the calculated pI to maintain a strong, stable net charge and avoid the precipitation risk that occurs right at the pI.
- When separating a mixture of proteins by ion exchange, compare each protein's charge-vs-pH profile to find a pH where your target protein's charge is clearly separated from contaminants.
- Treat the calculated pI as a starting estimate for heavily modified proteins, and confirm experimentally — for example, by running an isoelectric focusing gel — where precision matters.
- If your protein is not sequence-only (e.g. it is glycosylated or carries bound cofactors), cross-check the predicted pI against a literature or database value before relying on it for method design.
Common Mistakes to Avoid
- Forgetting post-translational modifications: phosphorylation, glycosylation, and acetylation can add or remove ionizable groups, shifting the true pI away from the sequence-based prediction.
- Including non-protein characters: leftover FASTA headers, line numbers, or non-standard characters in a pasted sequence can distort residue counts; always double-check the cleaned sequence length matches what you expect.
- Treating the pI as an exact experimental value: this is a theoretical estimate based on isolated side-chain pKa values; the protein's folded structure, local environment, and ionic strength can shift the true pI by up to a full pH unit in either direction.
- Running buffers too close to the predicted pI: operating within 1 pH unit of the pI risks precipitation or poor resolution; choose a buffer pH clearly above or below the calculated value.
Frequently Asked Questions
What is the difference between pI and net charge at pH 7?
The pI is the single pH value at which a protein has zero net charge, while the net charge at pH 7 tells you the charge the protein actually carries under near-physiological conditions. A protein can have a pI of 9.2 yet still carry a small positive charge at pH 7, since pH 7 sits below its pI. Knowing both values together is more useful than either alone: the pI predicts behavior in pH-gradient methods like isoelectric focusing, while the charge at pH 7 predicts how the protein will behave in standard aqueous buffers, lysates, or native gels run near physiological pH.
Why does my calculated pI differ slightly from the value listed on UniProt or ExPASy?
Different databases and tools use different sets of pKa values for the ionizable side chains, and even small shifts of a few tenths of a pH unit per residue can move the final pI by a noticeable amount. This calculator uses the commonly cited Lehninger/ExPASy ProtParam pKa scale, which is the same family of values used by most academic tools, but other software may use EMBOSS, Bjellqvist, or Sillero scales instead. Post-translational modifications, bound cofactors, and the protein's actual folded structure are also not accounted for by any sequence-based pI predictor, which is why all of these tools report a theoretical estimate rather than an experimentally measured pI.
Can this calculator predict the pI of a protein with disulfide bonds or modified residues?
No — this calculator, like all sequence-based pI predictors, only considers the standard ionizable groups on unmodified amino acids: the N-terminus, C-terminus, and the side chains of Asp, Glu, His, Cys, Tyr, Lys, and Arg. Disulfide bonds remove the free thiol charge contribution from cysteine, and post-translational modifications such as phosphorylation, glycosylation, or acetylation can add or remove ionizable groups entirely, shifting the true pI away from the predicted value. For proteins that are heavily modified, the calculated pI should be treated as a starting estimate to refine experimentally, for example by running an isoelectric focusing gel.
Why is my protein least soluble exactly at its isoelectric point?
At the pI, a protein's positive and negative charges balance to zero net charge, which removes the electrostatic repulsion that normally keeps protein molecules apart in solution. Without that repulsion, protein molecules can approach each other more closely and aggregate or precipitate, which is why solubility typically reaches a minimum at or very near the pI. This same principle is exploited deliberately in isoelectric precipitation methods used to purify or concentrate certain proteins, and it is also why storage and running buffers are usually chosen to sit at least 1–2 pH units away from a protein's pI.
How do I choose the right pH buffer for ion exchange chromatography using my calculated pI?
Once you know your protein's pI, the rule is straightforward: at a buffer pH above the pI, the protein carries a net negative charge and will bind to an anion exchange resin, while at a buffer pH below the pI, it carries a net positive charge and will bind to a cation exchange resin. Most protocols choose a working pH at least 1 pH unit away from the pI to ensure a strong, stable net charge and avoid the precipitation risk that occurs right at the pI. If you are purifying a mixture of proteins, comparing their individual pI and net-charge-vs-pH profiles using this calculator can help you pick a pH where your target protein's charge is clearly separated from contaminating proteins.