Does this use average or monoisotopic masses?

Both. Average masses (the default, same as ExPASy ProtParam) suit SDS-PAGE and general lab work. Monoisotopic masses use the most abundant isotope of each element and match high-resolution mass spectrometry of peptides. For MKVLWAALLVTFLAGSKATA the average mass is 2,091.58 Da and the monoisotopic mass 2,090.20 Da.

How is the theoretical pI calculated?

The net charge is summed from the N-terminus, C-terminus, and ionizable side chains (K, R, H, D, E, C, Y) using the Bjellqvist pK values, with residue-specific N-terminal pK values, and the pH where the charge is zero is found by bisection. This reproduces ExPASy Compute pI/Mw; for ubiquitin it gives 6.56. Real pI can shift with modifications and folding.

How do I calculate protein concentration from A280?

Use Beer–Lambert: concentration (M) = A280 ÷ (ε280 × path length in cm). In mg/mL, divide A280 by the A280 (0.1%) value, which is ε280 ÷ MW. For a protein with Abs 0.1% of 1.0, an A280 of 0.5 in a 1 cm cell is 0.5 mg/mL. Enter your A280 in the calculator to get mg/mL and µM.

Why is one water molecule added?

Each peptide bond releases one water, so residue masses are the amino acid masses minus water. The free N-terminal H and C-terminal OH together make up one water molecule, which is added back.

What single-letter codes are accepted?

The 20 standard amino acids plus U (selenocysteine) and O (pyrrolysine). Ambiguous codes such as B, Z, J, and X are reported and excluded because their mass is not defined.

Are disulfide bonds or post-translational modifications included?

Enter the number of disulfide bonds to subtract 2.016 Da each (2.0157 Da monoisotopic). Other modifications such as phosphorylation (+79.97 Da) or glycosylation must be added manually.

How is the extinction coefficient calculated?

ε280 = 5500 × Trp + 1490 × Tyr, plus 125 for each cystine when all cysteine pairs form disulfides (Pace et al., 1995, as used by ProtParam). A sequence without Trp or Tyr has an unreliable sequence-only estimate.

The grand average of hydropathy is the mean Kyte–Doolittle hydropathy value of all residues. Positive values indicate a more hydrophobic protein and negative values a more hydrophilic one.

Protein Molecular Weight Calculator

Paste a sequence or FASTA to get average or monoisotopic molecular weight, theoretical pI, net charge at pH 7, ε280 and A280 at 1 mg/mL, protein concentration from A280, GRAVY, and residue composition.

Amino Acid Sequence

Enter single-letter codes or paste FASTA (e.g. MKVLWAALLVTFLAGSKATA). FASTA header lines, spaces, digits, hyphens, and * are ignored.

Mass type

Disulfide bonds

Remove initiator Met

Molecular Weight Daltons (Da), average

Molecular Weight kiloDaltons (kDa)

Residue Count amino acids

Charge and hydropathy

Theoretical pI
Net charge pH 7.0
GRAVY
Aliphatic index

Theoretical absorbance at 280 nm

Sequence estimate for a 1 cm path. A280 (0.1%) assumes 1 g/L = 1 mg/mL protein.

All cysteines reduced

ε280 (M⁻¹ cm⁻¹)

A280 (0.1%):

Maximum cystines

ε280 (M⁻¹ cm⁻¹)

A280 (0.1%):

Concentration from a measured A280

A280 (1 cm path)

Use ε280 for

— mg/mL · — µM

Residue Composition

Residue	Code	Count	Residue Mass (Da)	Total Mass (Da)	% residue mass

Enter a sequence above and click Calculate to see the molecular weight.

Amino acid residue masses, pK and hydropathy

Code	Amino acid	Average (Da)	Monoisotopic (Da)	Side-chain pK	Hydropathy
A	Ala · Alanine	71.0788	71.03711	—	1.8
R	Arg · Arginine	156.1875	156.10111	12.00	-4.5
N	Asn · Asparagine	114.1038	114.04293	—	-3.5
D	Asp · Aspartate	115.0886	115.02694	4.05	-3.5
C	Cys · Cysteine	103.1388	103.00919	9.00	2.5
E	Glu · Glutamate	129.1155	129.04259	4.45	-3.5
Q	Gln · Glutamine	128.1307	128.05858	—	-3.5
G	Gly · Glycine	57.0519	57.02146	—	-0.4
H	His · Histidine	137.1411	137.05891	5.98	-3.2
I	Ile · Isoleucine	113.1594	113.08406	—	4.5
L	Leu · Leucine	113.1594	113.08406	—	3.8
K	Lys · Lysine	128.1741	128.09496	10.00	-3.9
M	Met · Methionine	131.1926	131.04049	—	1.9
F	Phe · Phenylalanine	147.1766	147.06841	—	2.8
P	Pro · Proline	97.1167	97.05276	—	-1.6
S	Ser · Serine	87.0782	87.03203	—	-0.8
T	Thr · Threonine	101.1051	101.04768	—	-0.7
W	Trp · Tryptophan	186.2132	186.07931	—	-0.9
Y	Tyr · Tyrosine	163.1760	163.06333	10.00	-1.3
V	Val · Valine	99.1326	99.06841	—	4.2
U	Sec · Selenocysteine	150.0388	150.95364	—	—
O	Pyl · Pyrrolysine	237.3018	237.14773	—	—

Residue masses are amino acid masses minus water (ExPASy). Add 18.01524 Da (average) or 18.01056 Da (monoisotopic) once per chain. Terminal pK values: N-terminus 7.5 (A 7.59, M 7.0, S 6.93, P 8.36, T 6.82, V 7.44, E 7.7), C-terminus 3.55. Hydropathy: Kyte–Doolittle.

Method, worked example, sources, and privacy

MW = Σ residue masses + water − 2.016 Da × disulfide bonds. For the 20-residue example MKVLWAALLVTFLAGSKATA, the residues sum to 2,073.56 Da; adding 18.02 Da of water gives 2,091.58 Da (average) or 2,090.20 Da monoisotopic. It has one Trp and no Tyr, so ε280 = 5,500 M⁻¹cm⁻¹ and A280 (0.1%) = 5,500 ÷ 2,091.58 = 2.630. Two lysines and no acidic residues give a theoretical pI of 10.00. These values match ExPASy ProtParam.

ε280 uses the coefficients of Pace et al. (1995). Masses and pI follow ExPASy Compute pI/Mw and ProtParam. FASTA header handling follows the UniProt FASTA format.

Privacy: the sequence is processed only in your browser; this calculator does not upload or store it.

Summary

The Protein Molecular Weight Calculator determines the mass of a protein or peptide from its single-letter sequence by summing residue masses and adding one water molecule for the free termini. It reports average mass (for SDS-PAGE and general lab use) or monoisotopic mass (for high-resolution mass spectrometry), can subtract disulfide bonds and the initiator methionine, and adds the theoretical isoelectric point (Bjellqvist pK set, as in ExPASy Compute pI/Mw), net charge, extinction coefficient, A280-based concentration, GRAVY, and aliphatic index.

How it works

Paste a plain amino acid sequence or FASTA. Lines beginning with > are treated as FASTA headers and excluded; spaces, digits, hyphens and * are ignored.
Choose average or monoisotopic residue masses, and optionally remove the initiator methionine or subtract disulfide bonds (2.016 Da each).
The tool sums the residue masses and adds one water molecule (18.01524 Da average, 18.01056 Da monoisotopic) for the free N- and C-termini.
Theoretical pI is found by bisection of the net charge using the Bjellqvist pK values, the same method as ExPASy Compute pI/Mw.
ε280 = 5500 × Trp + 1490 × Tyr (+ 125 per cystine). Enter a measured A280 to convert it to mg/mL and µM.

Use cases

Estimate the expected mass of a recombinant protein before SDS-PAGE or mass spectrometry.
Check a synthetic peptide against its monoisotopic mass from MS.
Convert a NanoDrop A280 reading into protein concentration in mg/mL and µM.
Choose a buffer pH away from the theoretical pI for ion-exchange chromatography.
Compare molecular weights and charge of protein variants or mutants.

Frequently Asked Questions

Last updated: 2026-10-09 · Reviewed by Nham Vu