Isoelectric Point Calculator

How to Use the Isoelectric Point Calculator

1. Paste your protein amino acid sequence in single-letter code into the text box, or upload a .txt / .fasta file.
2. Click Calculate Isoelectric Point.
3. The tool displays the pI, net charge at pH 7.0, and the full charge-vs-pH curve from pH 0 to 14.
4. Use the Charge at a Specific pH field to look up the net charge at any pH of your choice.

About the Isoelectric Point (pI)

The isoelectric point (pI) is the pH at which a protein carries zero net electrical charge. At the pI, the protein has minimal solubility and will not migrate in an electric field, which is the basis of isoelectric focusing (IEF) and 2D gel electrophoresis.

The pI is determined by the ionizable groups of the protein: the N-terminus, C-terminus, and the side chains of Asp (D), Glu (E), His (H), Cys (C), Tyr (Y), Lys (K), and Arg (R).

This calculator uses the widely cited pKa values from the Lehninger biochemistry textbook (similar to ExPASy ProtParam). The net charge at each pH is calculated using the Henderson-Hasselbalch equation for each ionizable group.

Practical Applications of pI

• Ion exchange chromatography: At pH above pI, the protein is negative → use anion exchange. Below pI, it is positive → use cation exchange.
• Isoelectric focusing (IEF): Proteins migrate to their pI in a pH gradient gel.
• Protein solubility: Proteins are least soluble at their pI due to minimal charge repulsion.
• Buffer selection: Running buffers should be at least 1–2 pH units away from the protein's pI to maintain solubility.