Isoelectric Point Calculator

How to Use the Isoelectric Point Calculator

1. Paste your protein's amino acid sequence in single-letter code into the text box, or upload a .txt / .fasta file. FASTA headers (lines starting with >) and whitespace are stripped automatically.
2. Click Calculate Isoelectric Point.
3. Review the calculated pI, the net charge at pH 7.0 and pH 7.4, and the full charge-vs-pH curve plotted from pH 0 to 14.
4. Use the Charge at a Specific pH field to look up the predicted net charge at any pH relevant to your experiment, such as a running buffer or storage condition.

The Formula: Henderson–Hasselbalch Charge Summation

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. For each basic group (N-terminus, His, Lys, Arg), the fractional positive charge at pH is 1 / (1 + 10^(pH − pKa)). For each acidic group (C-terminus, Asp, Glu, Cys, Tyr), the fractional negative charge is 1 / (1 + 10^(pKa − pH)). The net charge at a given pH is the sum of all positive contributions minus the sum of all negative contributions, and the calculator performs a binary search across pH 0–14 to find the pH where this net charge crosses zero — that pH is the pI. This calculator uses the Lehninger/ExPASy ProtParam pKa scale for each ionizable group.

When to Use This Calculator

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.

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.

Interpreting Your Results

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.

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.

Related Protein Tools