This protein concentration calculator applies the Beer–Lambert law at 280 nm: concentration = A280 ÷ (ε × path length), returning molarity and mg/mL from your protein’s extinction coefficient and molecular weight — the fastest honest quantitation in the protein lab.

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Beer–Lambert at 280 nm, With Its Confessions Attached

Proteins absorb at 280 nm almost entirely through tryptophan and tyrosine side chains, so a single absorbance reading prices the solution: c = A/(ε·l). The defaults model an antibody-scale protein — A280 0.550, ε 55,000 M⁻¹cm⁻¹, MW 66 kDa, 1 cm path → c = 1.00 × 10⁻⁵ M = 10.0 µM = 0.660 mg/mL. The ε is sequence-derivable, which is the method’s elegance: ε(280) ≈ 5,500 × (#Trp) + 1,490 × (#Tyr) + 125 × (#disulfides) M⁻¹cm⁻¹ (the Gill–von Hippel / ProtParam relation) — paste a sequence into ProtParam, take its ε, and this calculator’s answer needs no standard curve, no incubation, no dye lot. The confessions ride along in the result note because A280 lies in specific, knowable ways: nucleic acids absorb nearby (the A260/A280 ratio is the contamination polygraph — pure protein ~0.57, pure DNA ~1.8), buffers and detergents can absorb or scatter, and a protein with NO tryptophan (some small proteins, collagen-ish sequences) is nearly invisible at 280 regardless of concentration. Dye-binding assays (Bradford, BCA, Lowry) answer those cases by different chemistry; A280 answers in five seconds when its assumptions hold — the working scientist’s triage.

The dual output (mg/mL AND µM) is occupational: enzymologists dose in molarity (active sites are counted in moles), production and formulation teams spec in mg/mL (mass is what ships), and confusing the two across a 66 kDa protein is a 66,000-fold error genre all its own. MW comes from the sequence — our Protein Molecular Weight Calculator computes it from composition when you don’t have it at hand.

Worked Examples (Verified)

  • Defaults: 0.550/(55,000 × 1) = 10.0 µM → 0.660 mg/mL at 66 kDa.
  • The small-protein trap: a 12 kDa protein with ε 8,480 (few aromatics): A280 0.212 in 1 cm → 25.0 µM → 0.300 mg/mL — the same absorbance as a concentrated big protein would be a dilute small one; units discipline saves the experiment.
  • E0.1% shortcut: BSA’s E0.1% ≈ 0.667 → an A280 of 0.667 reads 1.00 mg/mL directly, no MW required — the handbook shortcut the note describes.

A280 Discipline

  • Blank against the EXACT buffer (DTT, imidazole and some detergents absorb at 280 — imidazole from His-tag elutions is the classic false-concentration culprit).
  • Stay in the linear range (~0.1–1.0 AU neat): dilute concentrated stocks into range and multiply back rather than trusting a 2.8 AU reading.
  • Warburg–Christian correction (protein mg/mL ≈ 1.55×A280 − 0.76×A260) rescues nucleic-acid-contaminated preps approximately when purification can’t.
  • ε depends on folding state slightly (ProtParam gives reduced vs oxidized cystine variants — pick the one matching your protein’s disulfides).
  • Aggregate/scattering shows as rising baseline toward 320–340 nm: check A320 ≈ 0 before believing A280; centrifuge/filter and re-read if not.

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Frequently Asked Questions

How do you calculate protein concentration from A280?

c = A280 ÷ (ε × path length), then mg/mL = c (mol/L) × MW (g/mol). Defaults: 0.550/(55,000 × 1 cm) = 10 µM, × 66,000 = 0.660 mg/mL. This calculator takes your ε, MW and path length and returns both unit systems with contamination checks.

Where do I get the extinction coefficient?

From the sequence: ε(280) ≈ 5,500 per tryptophan + 1,490 per tyrosine + 125 per disulfide bond (M⁻¹cm⁻¹) — tools like ExPASy ProtParam compute it from a pasted sequence, in reduced and oxidized variants. Handbook proteins have published values (BSA ε ≈ 43,824; IgG ≈ 210,000 for the intact ~150 kDa antibody, i.e., E0.1% ≈ 1.4). When no sequence exists, a calibrated dye assay against a BSA standard is the fallback.

What does A260/A280 tell me?

Purity: pure protein ≈ 0.57, pure DNA ≈ 1.8, pure RNA ≈ 2.0. A prep reading 1.2 is telling you nucleic acid is inflating A280 — purify further or apply a correction (Warburg–Christian) rather than reporting the flattering number. Note the ratio is a warning light, not a precise composition meter: it flags contamination, it doesn’t quantify the split exactly.

Why is my protein invisible at 280 nm?

No (or almost no) tryptophan/tyrosine: the chromophores ARE the aromatics. ProtParam will show ε near zero for such sequences (some structural proteins, small peptides). Use Bradford or BCA (which read peptide bonds and other residues), or monitor A205–220 nm peptide-bond absorbance with its own interferences. The method can’t fail more honestly than printing its assumption — ε — on the input form, as here.

A280 vs Bradford vs BCA — which assay when?

A280: purified protein, known sequence, instant, non-destructive (recover the sample) — the default for purified work. Bradford: fast, cheap, detergent-sensitive, protein-to-protein variable — good for crude lysates’ rough totals. BCA: detergent-tolerant, more uniform response, slower (incubation) — the lysate/quantitation workhorse. Choose by sample state, not habit; cross-check a new protein once by two methods.

What path length do microvolume instruments use?

NanoDrop-style pedestals compress the sample to ~0.05–0.1 cm physical paths and the software typically reports the 1-cm-equivalent absorbance. If your instrument reports raw short-path values, enter the true path here (0.1) — a 10× path error is a 10× concentration error wearing a lab coat. Cuvette users: 1 cm standard; 0.5 and 0.2 cm cuvettes exist for precious/concentrated samples.

Can I measure concentration in a mixture?

A280 gives TOTAL aromatic absorbance — in a mixture it returns a blended figure interpretable only if one species dominates or the others’ contributions are known and subtracted. True mixture quantitation needs separation (chromatography with peak integration) or species-specific assays (ELISA, activity assays). Gel densitometry (band intensity vs standards) is the bench approximation for known mixtures.

Is this protein concentration calculator free?

Yes — free, instant, no sign-up. Enter A280, extinction coefficient, molecular weight and path length for concentration in mg/mL and µM with purity checks.