Protein Molecular Weight Calculator
The molecular weight of a protein is the mass of one mole of the polypeptide, in daltons (Da) — numerically the same as g/mol. This calculator adds the average residue mass of every amino acid in your sequence and then adds back one water molecule (18.0153 Da) for the free ends of the chain: MW = Σ(n_residue × M_residue) + 18.0153. For the 76-residue protein ubiquitin that gives 8564.84 Da, or 8.565 kDa. It reports the mass in both Da and kDa, the residue composition it counted, and µg/nmol and nmol/µg so you can move straight between mass and moles. The number is for the unmodified chain exactly as you typed it — tags, signal peptides, glycosylation and bound cofactors are not included.
Almost every quantitative step with a purified protein needs its molecular weight: turning an A280 reading into a molar concentration, deciding how much to weigh for a stock, comparing a band on an SDS-PAGE gel to a predicted size, or working out a molar ratio for a binding assay. It is the bridge between the mass a balance or a spectrophotometer reports and the moles a reaction actually cares about. This page is part of the protein calculator set and it shows its working rather than handing back an opaque number — every residue mass is listed, the water correction is explicit, and the composition it counted is displayed so you can reproduce the result by hand.
The calculation rests on one idea: a residue inside a chain is lighter than the free amino acid, because forming each peptide bond releases a water molecule. So you cannot just add up free amino-acid masses. Instead you sum the residue masses — each amino acid minus one water — and then add a single water back at the end, because the two termini of the chain still carry their free α-amino hydrogen and α-carboxyl hydroxyl. For a chain of n residues that is n peptide-bond waters lost and one water restored, which is exactly what Σ(residue) + 18.0153 encodes.
The values used here are the average residue masses from Expasy (alanine 71.0788, glycine 57.0519, tryptophan 186.2132, and so on), which are weighted over the natural isotope distribution of each element. That makes the result match what an SDS-PAGE gel, a low-resolution mass spec, or a vendor datasheet reports as the "average" or "chemical" molecular weight. If you are reading a high-resolution MS peak you want the monoisotopic mass instead, built from the lightest isotope of each element — it is slightly lower and matters only for exact-mass work. For routine bench calculations the average mass is the right one.
Two things routinely make a predicted mass disagree with what you measure. First, the mature protein is often not the full translated chain: a cleaved signal peptide, a removed initiator methionine, or a processed pro-domain all subtract mass. Second, fusion tags and modifications add it — a hexahistidine (His6) tag contributes about 840.86 Da on its own, GST adds roughly 26 kDa, and glycosylation can add several kilodaltons of heterogeneous carbohydrate. This tool reports the chain you give it, so to predict a tagged construct include the tag residues in the sequence; to predict a mature protein give the mature sequence. The molecular weight then feeds directly into the protein concentration calculator, which converts an A280 reading to mg/mL, and pairs with the isoelectric point calculator for purification planning.
Protein Molecular Weight Calculator
How to use the protein molecular weight calculator
Paste your amino-acid sequence into the box using one-letter codes. Case is ignored, and spaces, line numbers and dashes from a FASTA paste are stripped automatically. The 20 standard residues are summed, plus the two mass-defined non-standard residues U (selenocysteine) and O (pyrrolysine).
Resolve any ambiguous codes before calculating. B (Asx), Z (Glx), X (unknown) and J have no single mass, so the calculator rejects them and tells you the position — replace each with the specific residue it stands for.
Press Calculate. The primary card is the molecular weight in Da; the cards below give kDa, the chain length, µg per nmol and nmol per µg, and the full residue composition that was counted.
Check the result against what you expect. If this is a tagged or processed construct, make sure the sequence you pasted matches the actual expressed protein — an initiator Met, a signal peptide or a cleavage site changes the answer.
Carry the mass into the protein concentration calculator to turn an A280 into mg/mL, or into the molarity calculator to work out how much to weigh or dilute for a target molar concentration.
Protein Molecular Weight formula
Variables and units
| Symbol | Meaning | Unit |
|---|---|---|
| MW | Molecular weight of the polypeptide | Da (g/mol) |
| M_residue | Average residue mass of each amino acid in the chain | Da |
| n_residue | Count of each residue in the sequence | — |
| 18.0153 | One water molecule added back for the free termini | Da |
| kDa | Kilodaltons (MW / 1000) | kDa |
| µg/nmol | Micrograms of protein per nanomole (= MW / 1000) | µg/nmol |
How protein molecular weight is calculated
- Clean the sequence: uppercase it and strip spaces, digits and dashes.
- Validate every character against the accepted alphabet; reject ambiguous codes (B/Z/X/J) with their position.
- Count each residue (the composition shown on the result card).
- Multiply every count by its average residue mass and sum the products.
- Add one water molecule, 18.0153 Da, for the free α-amino and α-carboxyl termini.
- Report the mass in Da and kDa, plus µg/nmol = MW/1000 and nmol/µg = 1000/MW.
Worked laboratory examples
Example 1 — Ubiquitin, a 76-residue protein
Given: sequence MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG · length 76 · composition includes L 9, I 7, K 7, T 7, G 6, E 6, Q 6, D 5
- Sum every residue count times its average mass across all 76 positions.
- The residue sum comes to 8546.83 Da.
- Add one terminal water: 8546.83 + 18.0153 = 8564.84.
- kDa = 8564.84 / 1000 = 8.565; µg/nmol = 8.565.
Result: MW = 8564.84 Da (8.565 kDa) — matching the published average mass of ubiquitin
Example 2 — A short peptide checked by hand
Given: sequence MKVLA · M 131.1926, K 128.1741, V 99.1326, L 113.1594, A 71.0788
- Residue sum: 131.1926 + 128.1741 + 99.1326 + 113.1594 + 71.0788 = 542.7375.
- Add one water: 542.7375 + 18.0153 = 560.7528.
- nmol/µg = 1000 / 560.7528 = 1.783.
Result: MW = 560.75 Da (0.561 kDa)
Example 3 — What a His6 tag adds
Given: a hexahistidine tag HHHHHH · H residue mass 137.1411
- Tag residues: 6 × 137.1411 = 822.8466.
- As a standalone peptide it would carry its own water: 822.8466 + 18.0153 = 840.86 Da.
- Fused to a protein the tag adds about 840.86 Da (0.84 kDa) before any linker or cleavage site — include the tag residues in the sequence to predict the construct.
Result: His6 tag ≈ 840.86 Da (≈0.84 kDa), excluding linker
Example 4 — From mass to moles for a stock
Given: ubiquitin, MW 8564.84 Da · you weigh out 2.0 mg and want a 100 µM stock
- nmol/µg = 1000 / 8564.84 = 0.1168 nmol per µg.
- 2.0 mg = 2000 µg → 2000 × 0.1168 = 233.5 nmol.
- For 100 µM (100 nmol/mL): volume = 233.5 / 100 = 2.34 mL.
Result: Dissolve 2.0 mg in 2.34 mL for a 100 µM stock
Want the reasoning behind the numbers? Read How to Calculate Protein Molecular Weight.
How to interpret the result
The primary number is the average mass of one mole of exactly the chain you entered. Because it uses average isotope masses it lines up with a gel or a datasheet, not with a high-resolution monoisotopic MS peak — if those differ by a dalton or two, that is the isotope convention, not an error.
The kDa figure is what you compare to a gel. A His-tagged 30 kDa protein migrating slightly above its predicted size is normal; a large unexplained gap usually means a modification, an uncleaved tag or a degradation product.
µg/nmol and nmol/µg are the practical outputs. Most bench questions are not "what is the mass" but "I have this many micrograms — how many nanomoles, and how much do I dilute to reach a molarity". Those conversions sit beside the mass so you never need a second tool.
The composition table makes the result auditable. If the counts do not match what you expected — a missing cysteine, an extra tag — the sequence you pasted was not the one you intended, and the mass is telling you so.
Assumptions
- The chain is unmodified: no phosphorylation, glycosylation, acetylation, lipidation or bound cofactors, all of which add mass.
- Average isotope masses are used, giving the chemical/average molecular weight rather than the monoisotopic mass.
- The sequence is the actual expressed protein — any signal peptide, initiator Met or pro-domain that is cleaved in the mature protein should already be removed.
- Residues U and O are counted at their average masses; no other non-standard residue is summed.
- The polypeptide is a single linear chain; quaternary structure (a multimer) is a whole-number multiple of the monomer mass.
Limitations
- Post-translational modifications and fusion tags are not added automatically — include tag residues in the sequence or add the modification mass yourself.
- The average mass differs from the monoisotopic mass used in high-resolution mass spectrometry.
- Ambiguous residue codes cannot be summed and are rejected; a sequence with unknown positions cannot be sized exactly.
- Bound water, salts of crystallisation and residual buffer in a dried pellet mean the weighed mass is not pure protein.
Common mistakes
- Adding free amino-acid masses instead of residue masses, which over-counts by one water per residue.
- Forgetting the single terminal water, which under-counts the chain by 18.0153 Da.
- Predicting the mass of the full translated sequence but comparing it to the mature, signal-peptide-cleaved protein on a gel.
- Omitting a His/GST/GFP tag from the sequence and then wondering why the construct runs heavier.
- Confusing average and monoisotopic mass when reconciling with a high-resolution MS value.
FAQ
What is the difference between Da, g/mol and kDa?
Why does the calculator add 18.0153 at the end?
Why is my predicted mass different from the band on my gel?
Average or monoisotopic mass — which should I use?
How do I include a His tag or other fusion?
How do I convert the mass to a molar concentration?
Scientific Sources
- Expasy FindMod — average and monoisotopic residue masses · Tier 1 source
- Expasy Compute pI/Mw — molecular weight tool and documentation · Tier 1 source
- CIAAW / NIST — standard atomic weights underlying the residue masses · Tier 1 source
External links open in a new tab and carry rel="nofollow". We cite primary references (NIST, NIH/NCBI, peer-reviewed papers) and established manufacturer technical documentation (NEB, Thermo Fisher, Promega, IDT). Calculated results are estimates; your protocol and manufacturer guidance take precedence.
Last reviewed: 2026-09 · Method version: 1.0