Isoelectric Point (pI) Calculator
Calculate isoelectric point (pI) of a protein from its net charge. At pI, net charge = 0. Enter acidic and basic amino acid counts to estimate pI. Essential for gel electrophoresis and protein purification.
🧪 Biochemistry📐 net charge = 0 at pH = pI🧬 Biology
Number of Asp+Glu (acidic, pKa~4)
Number of Lys+Arg (basic, pKa~10)
Number of His (pKa~6)
Please enter valid values.
Formula & Reference
| Variable | Symbol | Formula | Units |
|---|---|---|---|
| Isoelectric Point (pI) Calculator | — | net charge = 0 at pH = pI | pH units |
Step-by-Step Examples
Example 1
Acidic Protein
Protein with 15 Asp+Glu, 5 Lys+Arg, 1 His.
- More acidic than basic residues
- pI will be below 7 (acidic range)
- Estimated pI ~ 4.5-5.5
- Verify with ProtParam for sequence-specific pI
✓ pI ~ 4-5, acidic protein
Example 2
Basic Protein
Histone H4: rich in Arg and Lys (~25 basic, ~5 acidic).
- More basic residues than acidic
- pI ~ 11
- Histones are basic to bind negatively charged DNA
- Estimated pI > 10
✓ pI ~ 11, strongly basic protein
Example 3
Neutral Protein
GFP: 15 acidic, 13 basic residues.
- Nearly balanced charges
- pI ~ 6.0 (slightly acidic)
- Good for purification at pH 7.4 where protein is negatively charged
- Use anion exchange chromatography
✓ pI ~ 6.0 for GFP
Real-World Applications
Protein Purification
Ion exchange chromatography separates proteins by charge at pH above/below pI.
2D Gel Electrophoresis
IEF (isoelectric focusing) separates proteins by pI in first dimension, SDS-PAGE by size in second.
Protein Solubility
Proteins are least soluble at their pI. Avoid pI in buffers for stable protein solutions.
Antibody Engineering
Adjust antibody pI for improved pharmacokinetics and reduced immunogenicity.
Common Mistakes to Avoid
⚠️
Estimating pI without full sequence
This calculator gives rough estimates. For accurate pI, use ExPASy ProtParam with full amino acid sequence.
⚠️
Ignoring N- and C-terminal charges
The protein termini also carry charges (pKa ~8 for N-term, ~3.5 for C-term). These are often ignored in rough estimates.
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Thinking pI is constant
pI depends on protein conformation and solvent. Denatured proteins may have slightly different effective pI than native form.
Frequently Asked Questions
What is the isoelectric point? ▾
The pH at which a protein has zero net charge. Above pI: protein is negatively charged. Below pI: positively charged. At pI: zero charge and minimum solubility.
How is pI used in IEF? ▾
In isoelectric focusing, proteins migrate in a pH gradient until they reach their pI, where they stop moving. Proteins are focused at their pI.
What is 2D-PAGE? ▾
Two-dimensional PAGE separates proteins by pI (first dimension, IEF) and by size (second dimension, SDS-PAGE). Resolves hundreds of proteins simultaneously.
Why are proteins least soluble at pI? ▾
At pI, no net charge means no electrostatic repulsion between protein molecules, promoting aggregation. Adding NaCl or adjusting pH improves solubility.
What pKa values should I use? ▾
Standard pKa values: Asp 3.9, Glu 4.1, His 6.0, Cys 8.3, Tyr 10.1, Lys 10.5, Arg 12.5, N-term 8.0, C-term 3.1. Vary with local environment.