Why does my SDS-PAGE band migrate differently than the predicted MW?

SDS-PAGE estimates MW based on the assumption that all proteins bind SDS uniformly (1.4 g SDS per g protein). Aberrant migration occurs with: glycosylated proteins (run higher), highly charged proteins, proline-rich proteins, membrane proteins (bind more SDS), and very basic proteins. The predicted sequence MW is always correct; the gel migration is the approximation.

Should I include the signal peptide in MW calculation?

It depends on the mature form you're working with. If your protein is secreted and the signal peptide is cleaved, calculate MW without it. If you're expressing in E. coli without signal peptide processing, include it. Add the His-tag (6× His = 840 Da) or other fusion tags to the total.

How accurate is the extinction coefficient calculation?

The Pace method (based on Trp/Tyr/Cys content) is accurate to within 5-10% for most native proteins. Assumptions: all Cys form disulfide bonds (if unknown, calculate both with and without Cys contribution). Denatured proteins have slightly different ε₂₈₀ values. For maximum accuracy, measure experimentally by amino acid analysis.

What is the isoelectric point (pI)?

The pI is the pH where the protein has zero net charge. Below pI, the protein is positively charged; above pI, negatively charged. The pI determines behavior in ion exchange chromatography and isoelectric focusing. Computed pI assumes the protein is unfolded — actual pI may differ due to buried residues and post-translational modifications.

How do post-translational modifications affect MW?

Common PTMs: phosphorylation (+80 Da per site), glycosylation (+162 per hexose), acetylation (+42), ubiquitination (+8,565 per Ub), methionine oxidation (+16), disulfide bond formation (-2 Da per bond). For mass spectrometry, these shifts identify the modification type and site.

Can I calculate MW for proteins with non-standard amino acids?

This calculator covers the 20 standard amino acids. For selenocysteine (Sec, U, 150.04 Da) or pyrrolysine (Pyl, O, 255.31 Da), substitute the appropriate mass. For synthetic peptides with D-amino acids, the masses are identical to L-forms.

Protein Molecular Weight Calculator

Calculate protein molecular weight from amino acid sequence or composition. Covers residue weights, pI estimation, extinction coefficient, and charge at pH.

Protein Presets

Amino Acid Sequence (one-letter code)

Query pH (for net charge)

Molecular Weight

26,869.12 Da

26.87 kDa

Residues

238

Amino acids

Isoelectric Point (pI)

5.60

Zero net charge pH

Net Charge (pH 7)

-7.20

Negative (anionic)

ε₂₈₀ (with Cys-Cys)

22,015 M⁻¹cm⁻¹

1W, 11Y, 1 S-S

ε₀.₁% (A₂₈₀ 1 mg/mL)

8.193

Abs of 1 mg/mL, 1 cm path

Charge vs pH

Blue = positive charge | Red = negative charge | pI ≈ 5.6

Amino Acid Composition

AA	Name	Count	%	Mass contrib.
G	Glycine	22	9.2%	1,254 Da
K	Lysine	20	8.4%	2,562 Da
L	Leucine	19	8.0%	2,149 Da
D	Aspartate	18	7.6%	2,071 Da
V	Valine	17	7.1%	1,684 Da
E	Glutamate	16	6.7%	2,065 Da
T	Threonine	15	6.3%	1,516 Da
F	Phenylalanine	13	5.5%	1,912 Da
N	Asparagine	13	5.5%	1,483 Da
I	Isoleucine	12	5.0%	1,357 Da
S	Serine	11	4.6%	957 Da
Y	Tyrosine	11	4.6%	1,794 Da
P	Proline	10	4.2%	971 Da
H	Histidine	10	4.2%	1,371 Da
A	Alanine	8	3.4%	568 Da
Q	Glutamine	8	3.4%	1,024 Da
M	Methionine	6	2.5%	786 Da
R	Arginine	6	2.5%	937 Da
C	Cysteine	2	0.8%	206 Da
W	Tryptophan	1	0.4%	186 Da

Planning notes, formulas, and examples

About the Protein Molecular Weight Calculator

Protein molecular weight (MW) is one of the most fundamental properties in biochemistry. It determines migration on SDS-PAGE gels, elution volume in size exclusion chromatography, sedimentation in ultracentrifugation, and is essential for converting between mass concentration (mg/mL) and molar concentration (µM). Every protein experiment — from Western blot band identification to drug dosing — depends on knowing the molecular weight.

The molecular weight of a protein is calculated from its amino acid sequence by summing the residue weights of all amino acids and subtracting water (18.02 Da) lost at each peptide bond. The 20 standard amino acids have residue weights ranging from 57.02 Da (glycine) to 186.21 Da (tryptophan), with an average of about 110 Da. A 300-residue protein therefore has an approximate MW of ~33,000 Da (33 kDa).

This calculator computes exact MW from either a one-letter amino acid sequence or amino acid composition counts. Beyond MW, it estimates the theoretical isoelectric point (pI), molar extinction coefficient at 280 nm, and net charge at a given pH — all critical parameters derivable from sequence alone. These calculations replicate the functionality of ExPASy ProtParam, the gold-standard online tool for protein physicochemical properties.

When This Page Helps

Protein MW calculation is essential for SDS-PAGE gel interpretation, mass spectrometry data validation, buffer preparation, and converting between mass and molar concentrations. It gives the computed molecular weight directly from sequence input.

How to Use the Inputs

Enter a one-letter amino acid sequence (e.g. MSKGEELFT...)
Or use composition mode to enter residue counts manually
Review the calculated molecular weight in Da and kDa
Check the amino acid composition breakdown
View the estimated pI and extinction coefficient
Use presets for common marker proteins
Compare with SDS-PAGE standards for band identification

Formula used

MW = Σ(residue masses) + 18.02 (water for termini). Residue mass = amino acid MW - 18.02 (water lost per peptide bond). Extinction coefficient: ε₂₈₀ = nTrp × 5500 + nTyr × 1490 + nCys-Cys × 125 (M⁻¹cm⁻¹). Theoretical pI: calculated by iterating pH until net charge = 0.

Example Calculation

Result: MW = 26,899 Da (26.9 kDa)

GFP (238 amino acids): sum of all residue masses + terminal water = 26,899 Da. Contains 1 Trp, 11 Tyr, 2 Cys → ε₂₈₀ = 21,890 M⁻¹cm⁻¹.

Tips & Best Practices

Copy sequence directly from UniProt — this calculator ignores non-amino acid characters
For fusion proteins, concatenate the tag sequence with your protein sequence
Include initiator methionine unless it's been confirmed cleaved by N-terminal processing
Compare calculated MW with ESI-MS observed mass to verify protein identity
Membrane proteins in SDS-PAGE often run 10-20% lower than the calculated MW
A "kDa" on a Western blot is an approximation — always verify by mass spectrometry

Amino Acid Residue Mass Table

The 20 standard amino acids have these residue masses (monoisotopic): **Gly (G)** 57.02, **Ala (A)** 71.04, **Val (V)** 99.07, **Leu (L)** 113.08, **Ile (I)** 113.08, **Pro (P)** 97.05, **Phe (F)** 147.07, **Trp (W)** 186.08, **Met (M)** 131.04, **Ser (S)** 87.03, **Thr (T)** 101.05, **Cys (C)** 103.01, **Tyr (Y)** 163.06, **His (H)** 137.06, **Asp (D)** 115.03, **Glu (E)** 129.04, **Asn (N)** 114.04, **Gln (Q)** 128.06, **Lys (K)** 128.09, **Arg (R)** 156.10. These are average masses; monoisotopic masses (using most abundant isotope) differ slightly and are used in high-resolution mass spectrometry.

pI Estimation Algorithm

The theoretical pI is computed by iterating pH from 0 to 14 and calculating the net charge at each pH using the Henderson-Hasselbalch equation for each ionizable group. **pKa values used**: N-terminus = 9.69, C-terminus = 2.34, Asp (D) = 3.65, Glu (E) = 4.25, Cys (C) = 8.18, Tyr (Y) = 10.07, His (H) = 6.00, Lys (K) = 10.54, Arg (R) = 12.48. The pH where net charge crosses zero is the pI. Multiple pI calculators exist (ExPASy, IPC, Bjellqvist) and may give slightly different results depending on the pKa values used.

SDS-PAGE Molecular Weight Standards

Common unstained MW markers: **10 kDa** (Aprotinin), **15 kDa** (Lysozyme), **25 kDa**, **35 kDa**, **40 kDa**, **55 kDa**, **70 kDa** (BSA), **100 kDa**, **130 kDa**, **170 kDa**, **250 kDa**. For accurate MW estimation by gel migration, plot log(MW) vs relative mobility (Rf) for the standards and interpolate your unknown. The linear range depends on gel percentage: 15% for 10-60 kDa, 10% for 15-150 kDa, 7.5% for 30-300 kDa.

Sources & Methodology

Last updated: June 1, 2026

Frequently Asked Questions

SDS-PAGE estimates MW based on the assumption that all proteins bind SDS uniformly (1.4 g SDS per g protein). Aberrant migration occurs with: glycosylated proteins (run higher), highly charged proteins, proline-rich proteins, membrane proteins (bind more SDS), and very basic proteins. The predicted sequence MW is always correct; the gel migration is the approximation.