Protein Concentration Calculator

Three approaches: (1) Calculate from sequence using the Pace method (Trp × 5500 + Tyr × 1490 + Cystine × 125), available from ExPASy ProtParam, (2) Look up in literature or protein databases, (3) Determine experimentally by measuring A₂₈₀ of a sample with known concentration (from amino acid analysis or quantitative amino acid hydrolysis).

For mixtures, A₂₈₀ gives total protein concentration only if you use an average extinction coefficient. This is unreliable because different proteins have very different ε₂₈₀ values. For mixtures, colorimetric assays like BCA or Bradford provide more consistent results because they measure total peptide bonds or amino acids regardless of aromatic content.

NanoDrop spectrophotometers use 0.05-1 mm path lengths and require only 1-2 µL of sample. They internally correct to 1-cm equivalent readings. If using the raw absorbance from a NanoDrop, set path length to 0.1 cm (1 mm) or use the auto-corrected value with 1.0 cm. Short path lengths allow measurement of concentrated samples without dilution.

Common causes: (1) Air bubbles in the cuvette, (2) Particulates or aggregated protein scattering light, (3) Temperature fluctuations affecting buffer absorbance, (4) Dirty cuvette surfaces, (5) Instrument warm-up issues. Allow 15 minutes warm-up, blank with matching buffer, centrifuge samples before measurement, and use clean cuvettes.

Many common buffer components absorb at 280 nm: DTT (significant above 250 nm), imidazole (strong absorbance at 280 nm, problematic for His-tag purification eluates), nucleotides (ATP, GTP), Triton X-100 and NP-40 (aromatic rings). Always blank against the same buffer. Dialyze or desalt protein samples if buffer interference is suspected.

The Warburg-Christian method corrects for nucleic acid contamination using two wavelengths: Protein (mg/mL) = 1.55 × A₂₈₀ - 0.76 × A₂₆₀. This works because nucleic acids absorb more strongly at 260 nm than at 280 nm, while proteins show the opposite pattern. The correction is approximate and works best when contamination is less than 20%.

Yes: µM = (mg/mL × 1000) / MW(Da), or mg/mL = µM × MW(Da) / 1000. For example, 1 mg/mL of a 50 kDa protein = 1000/50000 × 1000 = 20 µM. This conversion requires knowing the molecular weight accurately. Many researchers use mg/mL for protein stocks and µM for binding experiments.

For standard 1-cm cuvettes, the practical detection limit is ~20-50 µg/mL (A₂₈₀ = 0.01-0.05 for typical proteins). NanoDrop instruments can measure 0.1-400 mg/mL with 1-2 µL. For sub-µg/mL concentrations, use fluorescence-based assays (Qubit, NanoOrange) or silver-stained gels for detection.

It depends on the application. Use molar concentration (µM, nM) for binding assays (Kd determination), enzyme kinetics (Km, kcat), and stoichiometric calculations. Use mass concentration (mg/mL) for formulation, column loading calculations, and when MW is uncertain (e.g., glycoproteins with heterogeneous modifications).