Protein Isoelectric Point Calculator
Calculate protein isoelectric point with our free science calculator. Uses standard scientific formulas with unit conversions and explanations.
Reviewed by Daniel Agrici, Founder & Lead Developer
Formula
Net charge = Sum[1/(1+10^(pH-pKa))] for basic groups - Sum[1/(1+10^(pKa-pH))] for acidic groups
The isoelectric point is the pH where the net charge equation equals zero. Basic groups (N-terminus, Lys, Arg, His) contribute positive charge; acidic groups (C-terminus, Asp, Glu, Cys, Tyr) contribute negative charge. A bisection algorithm finds the zero-crossing pH.
Worked Examples
Example 1: pI of a Basic Protein
Problem:Calculate the pI for the sequence MKRHHKKRM (9 aa) which is rich in basic residues.
Solution:Charged residues: K(3), R(2), H(2) = 7 positive; M(2) = 0 negative\nWith N-term (+) and C-term (-), the protein is heavily basic.\nBisection finds pH where positive charges from K,R,H equal terminal negative charge.\npI shifts far above 7 due to dominant basic residues.
Result:pI approximately 11.5 (highly basic protein migrates to cathode at physiological pH)
Example 2: pI of an Acidic Protein
Problem:Calculate the pI for MDDEEEDDM (9 aa) rich in acidic residues.
Solution:Charged residues: D(4), E(3) = 7 negative groups; no positive side chains.\nOnly N-terminal amino group provides positive charge.\npI must be very low for single positive charge to balance 7 negative groups.
Result:pI approximately 3.2 (highly acidic protein, suitable for anion exchange at pH 7)
Frequently Asked Questions
What is the isoelectric point (pI) of a protein?
The isoelectric point (pI) is the pH at which a protein carries no net electrical charge. At this pH, the sum of all positive charges from basic amino acids (Lys, Arg, His) and the N-terminus equals the sum of all negative charges from acidic amino acids (Asp, Glu, Cys, Tyr) and the C-terminus. Below the pI, the protein has a net positive charge and migrates toward the cathode in electrophoresis. Above the pI, it has a net negative charge and migrates toward the anode. The pI is critical for protein purification techniques like isoelectric focusing and ion exchange chromatography.
How is the isoelectric point calculated?
The pI is calculated using the Henderson-Hasselbalch equation applied to all ionizable groups in the protein. Each amino acid side chain and the terminal groups have characteristic pKa values. The net charge is computed as the sum of protonation states at a given pH using: charge = 1/(1+10^(pH-pKa)) for basic groups and -1/(1+10^(pKa-pH)) for acidic groups. A bisection algorithm iteratively finds the pH where total charge equals zero. Different pKa scales (EMBOSS, DTASelect, Solomon) give slightly different results because actual pKa values are influenced by the proteins three-dimensional structure.
Why is the pI important for protein purification?
The pI determines how a protein behaves in charge-based separation techniques. In ion exchange chromatography, proteins bind to charged resins when their net charge is opposite to the resin. Knowing the pI helps select the right pH for binding and elution buffers. In isoelectric focusing (IEF), proteins migrate through a pH gradient until they reach their pI where they stop. For 2D gel electrophoresis, IEF is the first dimension. The pI also affects protein solubility, which is typically lowest at the pI, a principle used in isoelectric precipitation for large-scale purification.
References
Reviewed by Daniel Agrici, Founder & Lead Developer · Editorial policy