What is the protein isoelectric point (pI) and why is it important?

The isoelectric point (pI) of a protein is the specific pH at which its net electrical charge equals zero. At pH values below its pI, a protein carries a net positive charge (cationic) because basic side chains are protonated. At pH values above its pI, it carries a net negative charge (anionic) because acidic side chains lose protons. The pI is essential for designing ion-exchange chromatography (IEX) purification protocols, 2D gel isoelectric focusing (IEF), crystallization screenings, and predicting solubility and colloidal stability.

How does the Bjellqvist / ExPASy pKa scale calculate theoretical pI?

The Bjellqvist scale (developed at ExPASy/Compute pI/MW) provides empirically calibrated pKa values for ionizable groups within polypeptide chains: N-terminus (9.69), C-terminus (3.55), Lysine (10.00), Arginine (12.00), Histidine (6.74), Aspartate (4.05), Glutamate (4.45), Cysteine (8.55), and Tyrosine (10.00). At any pH, the net charge is determined by summing the fractional protonation states of positive residues and deprotonation states of negative residues using the Henderson-Hasselbalch relation. A numerical bisection algorithm searches the pH domain (0.0 to 14.0) to find the precise root where net charge equals 0.00.

Why do proteins often precipitate or aggregate when buffer pH is close to their pI?

Proteins remain stably dispersed in aqueous solution largely due to electrostatic repulsion between molecules carrying like charges. When the ambient solution pH equals the protein's pI, net electrostatic charge drops to zero. Without mutual electrostatic repulsion, short-range attractive forces (such as hydrophobic interactions, van der Waals forces, and dipole-dipole attractions) dominate, causing protein molecules to self-associate and precipitate out of solution. Consequently, purification and formulation buffers are typically buffered at least 0.5 to 1.0 pH unit away from the target protein's pI.

How do post-translational modifications (PTMs) impact experimental pI?

Theoretical pI calculations assume an unmodified, linear polypeptide backbone. However, post-translational modifications (PTMs) frequently alter charge: phosphorylation adds dianionic phosphate groups (-2 charge at physiological pH), shifting the observed pI significantly toward acidic pH; acetylation of lysine side chains or the N-terminus neutralizes positive charge, also lowering the pI; and deamidation of asparagine/glutamine to aspartate/glutamate introduces new carboxylates. Disulfide bond formation can also slightly alter the apparent pKa of neighboring residues.

How is theoretical protein molecular weight calculated?

Protein molecular weight is calculated as the sum of the average isotopic masses of all amino acid residues in the sequence, plus 18.01524 Da corresponding to one molecule of water (representing the free terminal amino -H and carboxyl -OH groups restored upon peptide condensation). For example, a 100-residue polypeptide has 99 peptide bonds formed with the release of 99 water molecules, leaving two free termini. The average residue mass across standard proteomes is approximately 110–111 Da.

How is the molar extinction coefficient at 280 nm (ε280) determined?

Using the widely accepted Pace et al. (1995) / Gill and von Hippel method, the molar extinction coefficient at 280 nm in water or buffer is calculated from the aromatic amino acid composition: ε280 = (Trp count × 5,500) + (Tyr count × 1,490) + (Cystine pairs × 125) M⁻¹ cm⁻¹. Tryptophan and tyrosine absorb strongly in the near-UV (280 nm), whereas phenylalanine absorbs at 257 nm and has negligible contribution at 280 nm. This coefficient allows direct spectrophotometric quantification of protein concentration using Beer-Lambert's law (A280 = ε × l × c).

Protein Isoelectric Point (pI) & MW Calculator

Calculate protein isoelectric point (pI), molecular weight (MW in kDa), net charge at physiological pH, and extinction coefficient from amino acid or FASTA sequences.

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Isoelectric Point (pI)
Molecular Weight (kDa)
Sequence Length
Net Charge (pH 7.4)
Extinction Coeff

Bjellqvist / ExPASy Proteomics Scale

Protein Isoelectric Point (pI) & MW Calculator

Calculate protein isoelectric point (pI), molecular weight (MW in Da and kDa), net charge at physiological pH (7.0 and 7.4), and extinction coefficient at 280 nm using the Bjellqvist pKa scale and bisection root-finding algorithm. Supports raw single-letter amino acid sequences and standard multi-line FASTA formats.

Select Benchmark Protein Presets:

Protein Sequence (FASTA or Raw Amino Acid Code)

Paste single-letter amino acid codes (A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V). FASTA headers and whitespace are auto-cleaned.

147 residues (aa)

Theoretical Isoelectric Point

7.54pH

Net electrical charge = 0.00 at this pH. The protein exists as an electro-neutral zwitterion with minimal mutual electrostatic repulsion.

Domain: pH 0.0 – 14.0Algorithm: Numerical Bisection Root Search

Total Molecular Weight (MW)

16.00kDa

= 15,998 Da (includes terminal H₂O, 18.02 Da)

Average Residue Mass: 108.8 Da/aaSingle Chain Monomer

Sequence Length

147aa

Standard peptide chain

Net Charge @ pH 7.4Neutral

+0.46

Human physiological blood pH

Net Charge @ pH 7.0Cationic

+2.15

Neutral aqueous buffer

Extinction Coeff (ε280)

15,595M⁻¹ cm⁻¹

A280 (0.1%): 0.975 AU

Interactive Buffer pH Probe

Slide or type any target pH to evaluate real-time net charge and ion-exchange chromatography behavior.

Net Charge @ pH 7.4:+0.46

pH 1.0 (Strong Acid)pI = 7.54pH 13.0 (Strong Base)

Ion-Exchange Chromatography (IEX) Prediction for pH 7.4

Caution: Near Isoelectric Point (Net Charge ≈ 0)

At pH 7.4, the protein is virtually neutral (Net Charge = 0.46). It will show weak or negligible binding to both anion and cation exchange matrices, and has an increased tendency to precipitate.

To achieve reproducible chromatographic retention, adjust your binding buffer pH at least 1.0 unit above or below the pI (7.54).

Positively Charged (Basic)23Lys (K), Arg (R), His (H)

Negatively Charged (Acidic)15Asp (D), Glu (E)

Cysteines (Cys)2Potential disulfide bonds

Tyrosines (Tyr)3Near-UV A280 absorber

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Proteomics & Biochemistry Laboratory Guide

Understanding Protein Isoelectric Point (pI) & Molecular Weight

1What is the Isoelectric Point (pI)?

Every protein is a heteropolymer made up of ionizable and non-ionizable amino acids. Seven standard amino acids possess side-chain groups that can undergo reversible proton exchange: basic residues (Lysine, Arginine, Histidine), acidic residues (Aspartate, Glutamate), and weakly ionizable residues (Cysteine, Tyrosine).

The Isoelectric Point (pI) is defined as the exact solution pH at which the positive electrical charges exactly counterbalance the negative electrical charges, yielding a net charge of zero.

2The Bjellqvist / ExPASy pKa Scale

The standard pKa benchmark utilized by ExPASy (Compute pI/MW) is the Bjellqvist scale. Developed through extensive experimental measurements of 2D gel isoelectric focusing (IEF), it provides calibrated dissociation constants for peptide environments:

N-terminus: 9.69 | C-terminus: 3.55
Lys (K): 10.00 | Arg (R): 12.00 | His (H): 6.74
Asp (D): 4.05 | Glu (E): 4.45
Cys (C): 8.55 | Tyr (Y): 10.00

3Purification Strategy: Ion Exchange (IEX)

Ion exchange chromatography separates proteins according to surface charge. By setting buffer pH relative to the protein's pI:

pH < pI: Protein carries a net positive charge (cationic) and binds to Cation Exchange Resins (SP Sepharose, CM Cellulose).
pH > pI: Protein carries a net negative charge (anionic) and binds to Anion Exchange Resins (Q Sepharose, DEAE Cellulose).
Rule of Thumb: Maintain working pH at least 1.0 unit away from pI to ensure complete binding and avoid isoelectric precipitation.

4Molecular Weight & UV Extinction (A280)

Protein molecular weight equals the sum of standard residue masses plus 18.01524 Da (terminal H₂O).

Using the Pace et al. (1995) formula, the molar extinction coefficient at 280 nm is estimated from aromatic residues:
ε280 = (Trp × 5500) + (Tyr × 1490) + (Cystine × 125) M⁻¹ cm⁻¹
This coefficient allows direct concentration determination via Beer-Lambert's Law (A = ε · l · c).

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Protein Isoelectric Point (pI) & MW Calculator

Protein Isoelectric Point (pI) & MW Calculator

Next Steps with this file

Understanding Protein Isoelectric Point (pI) & Molecular Weight

1What is the Isoelectric Point (pI)?

2The Bjellqvist / ExPASy pKa Scale

3Purification Strategy: Ion Exchange (IEX)

4Molecular Weight & UV Extinction (A280)

Frequently Asked Questions

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Protein Isoelectric Point (pI) & MW Calculator

Next Steps with this file

Understanding Protein Isoelectric Point (pI) & Molecular Weight

1What is the Isoelectric Point (pI)?

2The Bjellqvist / ExPASy pKa Scale

3Purification Strategy: Ion Exchange (IEX)

4Molecular Weight & UV Extinction (A280)

Frequently Asked Questions