Estimate the isoelectric point of one amino acid or a whole sequence, then see its expected net charge at any pH you choose.
Advanced options
pKa choices
Termini (ends of the chain)
Display
How to use our Isoelectric Point Calculator
- Choose What are you calculating?, then enter either a Sequence (one-letter amino acid codes) or pick an Amino acid.
- Enter Compare at pH to check the estimated net charge in your buffer or sample condition.
- Open Advanced options if you want to change the pKa set or tell the calculator whether the Free N-terminus included? and Free C-terminus included? assumptions should stay on.
- Click Calculate, then read Estimated isoelectric point (pI) first and compare it with Overall charge at your pH to see whether the molecule should be positive, negative, or near neutral.
- Sanity-check the result: make sure Sequence length and Ionizable groups counted match what you expected, and read Note or warning if the sequence was cleaned or an assumption may affect accuracy.
Definitions
Isoelectric point (pI): The pH where a molecule's net charge is about zero.
Net charge: The total charge after adding the positive and negative contributions from all ionizable groups.
pKa: A number that tells how easily a group gains or loses a proton. Different pKa values change the estimated pI.
Ionizable groups counted: The side chains and terminal groups that can carry charge in this calculation, such as D, E, C, Y, H, K, R, plus free termini when included.
Free N-terminus: The amino end of the sequence, counted as a basic group when it is not chemically blocked.
Free C-terminus: The carboxyl end of the sequence, counted as an acidic group when it is not chemically blocked.
One-letter amino acid codes: Single capital letters like A, C, D, and K used to write peptide and protein sequences.
Common mistakes and quick fixes
Mistake: Pasting a protein name or words into Sequence (one-letter amino acid codes) instead of one-letter residue codes.
Fix: Use only standard one-letter amino acid letters, then confirm the cleaned Sequence length looks right.
Mistake: Leaving Compare at pH blank or typing a value outside 0 to 14.
Fix: Enter a real pH such as 7.0 so the calculator can show Estimated net charge at your pH and Overall charge at your pH .
Mistake: Using Single amino acid mode but expecting the Sequence (one-letter amino acid codes) box to affect the answer.
Fix: In that mode, choose the correct Amino acid ; the sequence box is ignored.
Mistake: Forgetting that blocked ends should change Free N-terminus included? or Free C-terminus included? .
Fix: If a terminus is chemically blocked, switch that setting to No so Estimated isoelectric point (pI) and Ionizable groups counted use the right assumptions.
Mistake: Comparing results from different tools without checking the pKa set .
Fix: Match the same pKa set and review Method used before deciding two results disagree.
Mistake: Treating a tiny nonzero Estimated net charge at your pH as a big chemical difference.
Fix: Read Overall charge at your pH for the plain-language summary and remember that values near 0 mean nearly neutral, not perfectly zero.
Limitations & Key Assumptions / Boundary Conditions
- This is an estimate based on published pKa values, so real measured pI can differ when a group's local environment shifts its pKa.
- Only the 20 standard amino acids are supported. Modified residues and post-translational modifications are not modeled.
- Sequence mode assumes the entered chain is a single peptide or protein and counts free termini only if you leave those settings turned on.
- Different pKa set choices can give different answers, especially for short peptides or borderline cases.
- The numeric search checks pH from 0 to 14 and reports the pH where net charge is closest to zero if there is no perfect zero crossing.
- Single amino acid mode follows the textbook bracketing idea around the neutral form, so it is best for standard classroom amino acids rather than unusual derivatives.
Methodology
How the calculator works
The tool estimates charge from each ionizable group, adds those charges together, and then finds the pH where the total is closest to zero. That pH is the estimated isoelectric point, or pI. Below the pI, a molecule tends to be more positive. Above the pI, it tends to be more negative [1].
Charge formulas used for sequences
q_acid = -1 / (1 + 10^(pKa - pH))
q_base = +1 / (1 + 10^(pH - pKa))
q_total(pH) = sum(q_base groups) + sum(q_acid groups)
Acidic groups include Asp, Glu, Cys, Tyr, and the free C-terminus when included. Basic groups include His, Lys, Arg, and the free N-terminus when included. The calculator uses the selected pKa set for those groups.
How pI is found
find pH in [0,14] where q_total(pH) = 0, or if no exact zero is found, use the pH where |q_total| is smallest
A stable numeric search checks the net charge across the standard aqueous pH range from 0 to 14 and returns the pH where the charge is closest to zero.
Single amino acid rule
pI = average of the two pKa values that bracket the neutral zwitterion form
For a neutral side-chain amino acid, this is usually the average of the alpha-carboxyl and alpha-amino pKa values. For acidic or basic side chains, use the two pKa values around the zero-charge form.
Mini-example
For aspartic acid in Single amino acid mode with a textbook pKa set, the neutral form lies between the alpha-carboxyl pKa and the side-chain carboxyl pKa. Using about 2.10 and 3.86 gives:
pI = (2.10 + 3.86) / 2 = 2.98
That is why aspartic acid has a low pI. At pH 7, the same amino acid is expected to have a negative net charge overall.
Assumptions used here
The model treats ionizable groups independently, ignores 3D structure effects, and does not model nonstandard residues or chemical modifications. For short peptides, terminal-group settings can noticeably change both Estimated isoelectric point (pI) and Estimated net charge at your pH.