Protein Isoelectric Point
Theoretical pI for one or more protein sequences, with optional fusion tags
Protein Sequence(s)

Raw sequence or multi-FASTA format. Input limit: 500,000 characters.

Options
to each sequence
💡 Quick Summary

Protein Isoelectric Point calculates the theoretical pI for one or more protein sequences. The pI is the pH at which the protein carries no net charge, which determines its migration on 2-D gels and its solubility behaviour. Common epitope and affinity tags can be added to each sequence before calculation.

📋 How to Use
  1. Paste one or more protein sequences (raw or FASTA) into the text area.
  2. Optionally add a fusion/epitope tag: choose the number of copies and the tag from the dropdowns.
  3. Choose a pK value set: EMBOSS (default) or DTASelect.
  4. Click Submit. Results appear as a table with Name, Length, and pI.
  5. Click Load Example to analyse two sample sequences.
  6. Use Copy All to copy the tabular results to your clipboard.
🧮 Formulas & Logic
Net charge
Q(pH) = PC(N-term) + K·PC(K) + R·PC(R) + H·PC(H) − D·PC(D) − E·PC(E) − C·PC(C) − Y·PC(Y) − PC(C-term)
Partial charge
PC(pK, pH) = 10^(pK−pH) / (10^(pK−pH) + 1) for basic groups; PC(pH, pK) = 10^(pH−pK) / (10^(pH−pK) + 1) for acidic groups
pI
pH at which Q(pH) = 0, found by bisection starting at pH 7.0 with step 3.5, halved each iteration.
📊 Result Interpretation
pI < 7

Acidic protein — net negative charge at physiological pH. Common in cytoplasmic proteins.

pI > 7

Basic protein — net positive charge at physiological pH. Common in DNA-binding and ribosomal proteins.

pI ≈ 7

Near-neutral protein. Will precipitate close to physiological pH.

EMBOSS pK

Recommended default. Based on widely used values from the EMBOSS suite.

DTASelect pK

Alternative set used by some mass-spectrometry workflows.

🔬 Applications
  • Predicting where a protein will migrate on a 2-D gel
  • Estimating protein solubility and precipitation behaviour
  • Comparing pI of wild-type vs. mutant protein variants
  • Designing purification strategies based on charge (e.g. ion-exchange conditions)
  • Evaluating the effect of common epitope tags (His6, FLAG, HA, etc.) on pI
⚠️ Common Mistakes & Warnings
Theoretical estimate only

The calculated pI assumes a fully unfolded protein and does not account for buried ionisable residues, post-translational modifications, or disulfide bonds.

Only standard amino acids counted

Non-standard characters (B, Z, X, *, gaps) are stripped before calculation. Only K, R, H, D, E, C, Y and the N- and C-termini contribute to net charge.

Tag length is included in reported length

When a fusion tag is added, the reported sequence length includes the appended tag residues.

❓ Frequently Asked Questions

Which pK set should I use?
EMBOSS is the recommended default and is the most widely used. DTASelect uses slightly different values derived from mass spectrometry data. Results differ by at most 0.1–0.5 pH units for most proteins.
Why does my protein pI differ from a database value?
Different tools use different pK values and may handle terminal residues differently. This tool uses the same algorithm as the original Sequence Manipulation Suite (EMBOSS set). Small differences of ±0.5 units are normal between tools.
What do the acidic (orange) and basic (blue) colours mean in the table?
Orange indicates a pI below 6.0 (acidic protein); blue indicates a pI above 8.0 (basic protein). Neutral proteins (pI 6–8) are shown in the default text colour.
Protein Isoelectric Point — logic ported from the Sequence Manipulation Suite (Paul Stothard, University of Alberta).