Isoelectric Point (pI) & Net Charge Calculator

Calculate pI and Net Charge from Amino Acid Sequence

Enter your protein or peptide sequence to calculate its isoelectric point (pI) and net charge at different pH values. Supports custom pKa values and advanced options.

Example Calculations

Try these sample protein or peptide sequences to see how the isoelectric point calculator works.

Hemoglobin Alpha Chain (1-20)

Protein

First 20 residues of human hemoglobin alpha chain.

Sequence: VLSPADKTNVKAAWGKVGAH

pKa Set: Standard (Lehninger)

pH Range: 0 - 14 (step 0.01)

Synthetic Peptide

Peptide

Short synthetic peptide with acidic and basic residues.

Sequence: ACDEHKR

pKa Set: Standard (Lehninger)

pH Range: 0 - 14 (step 0.01)

Custom pKa Example

Custom pKa

Peptide with user-defined pKa values.

Sequence: DEHKR

pKa Set: Custom

Custom pKa: 9.5,2.2,4.0,4.5,8.0,10.0,6.2,10.4,12.0

pH Range: 0 - 14 (step 0.01)

Insulin B Chain (1-15)

Protein

First 15 residues of human insulin B chain.

Sequence: FVNQHLCGSHLVEAL

pKa Set: Standard (Lehninger)

pH Range: 0 - 14 (step 0.01)

Other Titles
Understanding Isoelectric Point (pI): A Comprehensive Guide
Master protein and peptide charge analysis with pI and net charge calculations.

What is the Isoelectric Point (pI)?

  • Definition and Importance
  • Amino Acid Ionization
  • Protein Purification
The isoelectric point (pI) is the pH at which a molecule, such as a protein or peptide, carries no net electrical charge. It is a critical property for understanding protein solubility, stability, and behavior in different environments.
Amino Acid Ionization and pI
Amino acids have ionizable groups (N-terminus, C-terminus, and side chains) that gain or lose protons depending on pH. The pI is determined by the pKa values of these groups and the sequence composition.
Applications in Protein Purification
Knowing the pI is essential for techniques like isoelectric focusing, electrophoresis, and chromatography, where separation is based on charge differences.

pI in Practice

  • Hemoglobin pI is ~6.8, useful for blood analysis.
  • Peptide pI helps in designing purification protocols.

Step-by-Step Guide to Using the Isoelectric Point Calculator

  • Input Sequence
  • Select pKa Set
  • Interpret Results
Enter your amino acid sequence using single-letter codes. Choose the pKa set or enter custom values. Set the pH range and step for calculation. The calculator will compute the pI and net charge table.
Inputting the Sequence
Use only standard amino acid codes (ACDEFGHIKLMNPQRSTVWY). Non-standard residues are ignored or flagged as errors.
Selecting pKa Values
Standard pKa values are suitable for most cases. For special conditions or modified residues, use the custom option to enter your own pKa values.
Interpreting Results
The results include the calculated pI, net charge at pI, a table of net charge vs. pH, and the pKa values used. Use these for protein analysis, purification, or research.

Step-by-Step Example

  • Sequence: ACDEHKR, pKa set: standard, pI ≈ 7.1
  • Custom pKa for modified peptides.

Real-World Applications of Isoelectric Point Calculations

  • Protein Purification
  • Biopharmaceuticals
  • Food Science
Isoelectric point calculations are used in protein purification, formulation of biopharmaceuticals, and food science. pI helps predict solubility, aggregation, and stability of proteins in various environments.
Protein Purification and Analysis
Techniques like isoelectric focusing and ion-exchange chromatography rely on pI differences to separate proteins.
Biopharmaceutical Formulation
pI is used to optimize buffer conditions and prevent aggregation in therapeutic proteins.
Food Science and Nutrition
pI is important in dairy, egg, and plant protein processing, affecting texture and solubility.

Application Examples

  • Isoelectric focusing for protein separation.
  • Formulating stable protein drugs.

Common Misconceptions and Correct Methods

  • Ignoring Side Chains
  • Assuming All pKa are Equal
  • Overlooking Sequence Context
A common mistake is to ignore the contribution of side chain pKa values or to assume all pKa values are the same. Sequence context and environment can shift pKa values, affecting the pI calculation.
Side Chain Contributions
Only certain amino acids (D, E, C, Y, H, K, R) have ionizable side chains that affect pI. Their abundance and position matter.
Sequence and Environment Effects
pKa values can shift due to neighboring residues or solution conditions. Use custom pKa values if needed for accuracy.
Correct Calculation Methods
The calculator uses the Henderson-Hasselbalch equation and an iterative search to find the pH where net charge is zero.

Best Practice Guidelines

  • Don't ignore histidine (H) side chain.
  • Adjust pKa for post-translational modifications.

Mathematical Derivation and Examples

  • Henderson-Hasselbalch Equation
  • Iterative pI Search
  • Worked Example
The net charge at a given pH is calculated using the Henderson-Hasselbalch equation for each ionizable group. The pI is found by searching for the pH where the net charge crosses zero.
Henderson-Hasselbalch Equation
For acidic groups: charge = -1 / (1 + 10^(pKa - pH)). For basic groups: charge = +1 / (1 + 10^(pH - pKa)). The total net charge is the sum over all groups.
Iterative Search for pI
The calculator uses a bisection or grid search algorithm to find the pH where the net charge is closest to zero (the isoelectric point).
Worked Example
For the sequence ACDEHKR, the calculator computes the net charge at each pH and finds the pI where the net charge is zero. This is typically between 6 and 8 for most peptides.

Calculation Examples

  • Calculate pI for ACDEHKR using standard pKa values.
  • Use custom pKa for modified peptides.