How is protein molecular weight calculated from the sequence?

You add up the residue mass of every amino acid in the chain and then add one water molecule. Each amino acid's residue mass is the free amino acid minus a water, because forming each peptide bond releases a water. Adding one water back at the end accounts for the free amino and carboxyl groups at the two ends of the finished protein. Using average isotopic masses, this gives the average molecular weight in daltons.

What is the 280 nm extinction coefficient and where do 5500 and 1490 come from?

The molar extinction coefficient at 280 nm predicts how strongly a protein absorbs UV light, and it comes almost entirely from aromatic side chains. Using the Pace/Gill/Edelhoch method, each tryptophan contributes about 5500 M-1 cm-1, each tyrosine about 1490, and each disulfide bond (cystine) about 125 when cysteines are oxidized. A protein with no Trp or Tyr absorbs very little at 280 nm, which is why those proteins are hard to quantify by A280.

How do I turn an A280 reading into a protein concentration?

Divide the extinction coefficient by the molecular weight to get the absorbance of a 1 mg/mL solution (often written as A 0.1% or E1%). Then concentration in mg/mL equals your measured A280 divided by that value. For example, if 1 mg/mL gives an A280 of 1.5 and you read 0.75, the sample is 0.5 mg/mL. This is the number a NanoDrop uses when you tell it a protein's extinction coefficient.

What is theoretical pI and why does the calculated value vary between tools?

The theoretical isoelectric point (pI) is the pH at which the protein carries no net charge. It is found by summing the charge of every ionizable group (the N-terminus, C-terminus, and the side chains of Asp, Glu, Cys, Tyr, His, Lys, and Arg) across pH and locating where the total crosses zero. Different calculators use slightly different pKa tables, so the same sequence can give a pI that differs by a few tenths of a unit. Treat it as an estimate, not an exact measurement.

Should I use the reduced or oxidized extinction coefficient?

Use the reduced value if your cysteines are free thiols, which is the case for most cytoplasmic proteins and for samples run under reducing conditions. Use the oxidized value if the cysteines form disulfide bonds, common in secreted and extracellular proteins. The two differ only by 125 per disulfide bond, so for proteins with few or no cysteines the choice barely matters.

Protein Molecular Weight & Extinction Coefficient Calculator, Free

Amino acid sequence (one-letter code — spaces, numbers, and FASTA header lines are ignored)

Molecular weight

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Extinction coeff. (280 nm)

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A₂₈₀ of 1 mg/mL

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Theoretical pI

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Amino acid composition

How the numbers work

A protein is a chain of amino acids joined by peptide bonds, and every peptide bond forms by removing one water. So the mass of the chain is the sum of each amino acid's residue mass (the amino acid minus a water) plus a single water added back for the two free ends. The molecular weight here uses average isotopic masses, so it matches what you would report for a purified protein.

The extinction coefficient at 280 nm comes almost entirely from aromatic side chains. Using the Pace/Gill/Edelhoch method, each tryptophan adds about 5500 M^-1 cm^-1, each tyrosine about 1490, and each disulfide bond about 125 when cysteines are oxidized. Dividing that by the molecular weight gives the absorbance of a 1 mg/mL solution, so a spectrophotometer reading at 280 nm converts straight into concentration.

The theoretical pI is the pH where the summed charge of the N-terminus, C-terminus, and the ionizable side chains (Asp, Glu, Cys, Tyr, His, Lys, Arg) crosses zero. Different pKa tables shift it by a few tenths, so read it as an estimate.

Turning an A₂₈₀ into a concentration

The whole point of the extinction coefficient is quantification. Once you have the A₂₈₀ of a 1 mg/mL solution (call it the reference), any reading converts in one step:

concentration (mg/mL) = measured A280 ÷ (A280 of 1 mg/mL) Example — a protein whose 1 mg/mL solution reads A280 = 1.50: you measure A280 = 0.75 concentration = 0.75 / 1.50 = 0.50 mg/mL

This is exactly what a NanoDrop does when you enter a protein's extinction coefficient instead of using the generic "1 Abs = 1 mg/mL" setting.

FAQ

How is molecular weight calculated?
Sum every residue mass (amino acid minus a water) and add one water for the free ends. Average isotopic masses give the average MW in daltons.

Where do 5500 and 1490 come from?
They are the per-residue absorptivities at 280 nm (Pace/Gill/Edelhoch): 5500 M^-1 cm^-1 per Trp, 1490 per Tyr, plus 125 per disulfide bond when oxidized. Proteins with no Trp or Tyr barely absorb at 280 nm.

Reduced vs oxidized extinction coefficient?
Reduced = free cysteine thiols (most intracellular proteins). Oxidized = disulfide-bonded cysteines (many secreted proteins). They differ only by 125 per disulfide, so with few cysteines it hardly matters.

Why does the pI differ from another calculator?
Different pKa tables. The same sequence can shift by a few tenths of a pH unit. It is a theoretical estimate, not a measured value.

Does this handle a FASTA sequence?
Yes — a leading header line starting with ">" is ignored, and so are spaces, line breaks, and residue numbers. Non-standard letters (X, B, Z, U) are skipped and flagged.

Amino acid composition

How the numbers work

Turning an A280 into a concentration

FAQ

Turning an A₂₈₀ into a concentration