Protein & Peptide pI Calculator
Paste a one-letter amino acid sequence to estimate isoelectric point (pI) and net charge. Non-letter characters are ignored.
Estimated pI: -
Net Charge at selected pH: -
Classification: -
What is an isoelectric point (pI)?
The isoelectric point of a molecule is the pH where its total net charge is approximately zero. For proteins and peptides, pI depends on all ionizable groups: the N-terminus, C-terminus, and side chains (especially Asp, Glu, His, Cys, Tyr, Lys, and Arg).
At pH values below pI, proteins are typically more positively charged. At pH values above pI, they are typically more negatively charged. This matters in electrophoresis, ion-exchange chromatography, solubility optimization, and formulation work.
How this calculator works
This tool uses a common Henderson–Hasselbalch approach with representative pKa values to estimate net charge as a function of pH. It then applies a numerical bisection method from pH 0 to 14 to find the point where net charge crosses zero.
Ionizable groups included
- N-terminus (basic)
- C-terminus (acidic)
- Basic side chains: Lys (K), Arg (R), His (H)
- Acidic side chains: Asp (D), Glu (E), Cys (C), Tyr (Y)
How to use the calculator
- Enter a peptide/protein sequence in one-letter code.
- Set a pH if you want net charge at a specific condition (for example, pH 7.4).
- Click Calculate pI to get results instantly.
- Use Load Example to test with a known sequence format.
Why pI is useful in practice
1) Protein purification
If your buffer pH is near the target's pI, the protein may have minimal net charge and lower solubility. Moving pH away from pI often improves solubility and can improve chromatographic behavior.
2) Electrophoresis and focusing
In isoelectric focusing, proteins migrate until they reach local pH = pI. Accurate pI estimates help design gradients and interpret band patterns.
3) Formulation and stability
Charge state can influence aggregation, viscosity, and intermolecular interactions. pI gives a practical first estimate when screening buffer systems.
Important limitations
This is an estimate. Real pI values can shift due to local structure, neighboring residues, post-translational modifications, salt concentration, temperature, and experimental method. Use this as a fast planning tool, then confirm experimentally.
Quick FAQ
Does sequence length matter?
Yes. More ionizable residues generally increase charge complexity and can shift pI significantly.
Can I use modified residues?
Not directly in this simple tool. It only supports the 20 standard amino acids.
Why does my lab value differ?
Different pKa sets and experimental conditions can produce different results. That is normal.