Protein Molecular Weight
SDS-PAGE, Da/kDa. Sum of residue masses minus water. PTM, ε₂₈₀, pI from sequence.
Why This Biology Metric Matters
Why: MW needed for molarity, gel interpretation, and structural biology. SDS-PAGE estimates size.
How: Sum residue masses − (n−1)×18 for water loss. PTM add mass. ε₂₈₀ from Trp/Tyr. pI from charged residues.
- ●Average residue ~110 Da. 100 aa ≈ 11 kDa. Glycine smallest.
- ●SDS-PAGE: log(MW) vs mobility. Reducing vs non-reducing.
- ●ProtParam, ExPASy for automated calculation.
Protein Molecular Weight Calculator
From amino acid sequence — MW, ε₂₈₀, pI, composition. PTM support.
Sample Scenarios — Click to Load
Inputs
Post-Translational Modifications
No modifications added
⚠️For educational use only. Always confirm dosages and care with a licensed veterinarian.
🧬 Biology Facts
MW = Σ(residue masses) − (n−1)×18. Water lost in peptide bonds.
— Formula
ε₂₈₀ from Trp (5,500), Tyr (1,490), Cys (125) M⁻¹cm⁻¹.
— Extinction
SDS-PAGE: denaturing, size-based. kDa from ladder.
— SDS-PAGE
pI: pH where net charge zero. From Asp, Glu, His, Lys, Arg.
— pI
What is Protein Molecular Weight?
Protein molecular weight is the total mass of a protein molecule, calculated from its amino acid sequence. It's a fundamental property used in protein characterization, purification, and analysis. The molecular weight accounts for the sum of all amino acid residues minus water molecules lost during peptide bond formation.
Sequence-Based
Calculated directly from amino acid sequence using standard molecular weights.
Modifications
Accounts for post-translational modifications like phosphorylation, glycosylation, and acetylation.
Additional Properties
Also calculates extinction coefficient, isoelectric point, and amino acid composition.
How to Calculate Protein Molecular Weight
Step-by-Step Process
- Enter Amino Acid Sequence: Input the protein sequence using single-letter codes (A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V).
- Sum Amino Acid Weights: Add the molecular weight of each amino acid residue in the sequence.
- Add Modifications: Include any post-translational modifications (phosphorylation, acetylation, etc.) and their mass contributions.
- Subtract Water Loss: For each peptide bond formed, one water molecule (18.015 Da) is lost. Subtract (n-1) × 18.015 Da for n amino acids.
- Calculate Final MW: The final molecular weight is the sum of amino acids and modifications minus water loss.
Key Considerations
- Uses average molecular weights; monoisotopic differs for mass spec
- Water loss (n-1)×18.015 Da for peptide bonds
- Extinction coefficient from Trp, Tyr, Cys counts
- pI is approximate; accurate pI needs iterative pKa calculation
When to Use Protein Molecular Weight Calculation
🔬 Protein Purification
Determine expected molecular weight for SDS-PAGE analysis and gel filtration chromatography.
📐 Mass Spectrometry
Compare calculated MW with experimental mass spectrometry data to verify protein identity.
🧪 Concentration Determination
Calculate protein concentration using UV absorbance and extinction coefficient.
⚖️ Stoichiometry
Determine molar ratios in protein complexes and calculate reaction stoichiometry.
Formulas and Calculations
Molecular Weight Formula
• Σ(amino_acid_weights) = Sum of all amino acid molecular weights
• Σ(modifications) = Sum of post-translational modification weights
• n = Number of amino acids
• 18.015 = Molecular weight of water (lost during peptide bond formation)
Extinction Coefficient (280nm)
• nTrp = Number of tryptophan residues
• nTyr = Number of tyrosine residues
• nCys = Number of cysteine residues (disulfide bonds)
Amino Acid Molecular Weights
| Code | Amino Acid | Weight (Da) | Category |
|---|---|---|---|
| A | Alanine | 89.094 | Non-polar aliphatic |
| R | Arginine | 174.203 | Basic |
| N | Asparagine | 132.119 | Polar uncharged |
| D | Aspartic acid | 133.104 | Acidic |
| C | Cysteine | 121.154 | Polar uncharged |
| Q | Glutamine | 146.146 | Polar uncharged |
| E | Glutamic acid | 147.130 | Acidic |
| G | Glycine | 75.067 | Non-polar aliphatic |
| H | Histidine | 155.156 | Basic |
| I | Isoleucine | 131.175 | Non-polar aliphatic |
| L | Leucine | 131.175 | Non-polar aliphatic |
| K | Lysine | 146.189 | Basic |
| M | Methionine | 149.208 | Non-polar aliphatic |
| F | Phenylalanine | 165.192 | Aromatic |
| P | Proline | 115.132 | Non-polar aliphatic |
| S | Serine | 105.093 | Polar uncharged |
| T | Threonine | 119.119 | Polar uncharged |
| W | Tryptophan | 204.228 | Aromatic |
| Y | Tyrosine | 181.191 | Aromatic |
| V | Valine | 131.175 | Non-polar aliphatic |
Common Post-Translational Modifications
| Modification | Mass Change (Da) | Description |
|---|---|---|
| Phosphorylation | 79.966 | Addition of phosphate group (common on Ser, Thr, Tyr) |
| Acetylation | 42.011 | N-terminal or lysine acetylation |
| Methylation | 14.016 | Mono-methylation (can be di- or tri-methylation) |
| N-linked Glycosylation | 203.079 | N-linked glycosylation (Asn) |
| Ubiquitination | 8565.800 | Covalent attachment of ubiquitin (Lys) |
| SUMOylation | 11163.000 | Covalent attachment of SUMO (Lys) |
| Hydroxylation | 15.999 | Hydroxylation (common on Pro, Lys) |
| Palmitoylation | 238.230 | S-palmitoylation (Cys) |
| Myristoylation | 210.198 | N-myristoylation (Gly) |
| Farnesylation | 204.198 | C-terminal farnesylation (Cys) |
Best Practices
- Use single-letter codes; spaces are stripped automatically
- Verify sequence before calculation—invalid chars rejected
- Add PTMs for phosphorylated, acetylated, or glycosylated proteins
- Compare calculated MW with SDS-PAGE or mass spec for validation
Frequently Asked Questions
What is the difference between average and monoisotopic molecular weight?
Average molecular weight uses the weighted average of naturally occurring isotopes (used here). Monoisotopic molecular weight uses only the most abundant isotope of each element. Average MW is more commonly used for proteins, while monoisotopic MW is used in high-resolution mass spectrometry.
Why do we subtract water molecules?
During peptide bond formation, a condensation reaction occurs where a water molecule is released. For n amino acids, (n-1) peptide bonds are formed, so (n-1) water molecules are lost. This water loss must be subtracted to get the correct molecular weight of the protein.
How accurate is the calculated molecular weight?
The calculated molecular weight is accurate for the amino acid sequence, but actual experimental values may differ due to post-translational modifications, protein folding, or experimental error. Always verify with experimental methods like mass spectrometry.
What is extinction coefficient used for?
Extinction coefficient (ε) at 280nm is used to determine protein concentration using UV absorbance. The Beer-Lambert law: A = ε × c × l, where A is absorbance, c is concentration, and l is pathlength. Tryptophan, tyrosine, and cysteine are the main contributors to UV absorbance at 280nm.
How is isoelectric point (pI) calculated?
Isoelectric point is the pH at which a protein has no net charge. It's calculated by finding the pH where positive and negative charges balance. The calculator provides an estimate based on charged residues (Arg, Lys, His, Asp, Glu). Accurate pI requires iterative calculation considering all pKa values.