Protein concentration from A280

Worked example

• Human insulin precursor (UniProt P01308, 110 residues): 2 Trp, 4 Tyr and 6 Cys give ε280 = 2 × 5,500 + 4 × 1,490 + 3 × 125 = 17,335 M⁻¹cm⁻¹ with all cysteines paired, 16,960 reduced; the molecular weight is 11,980.9 Da.
• A280 = 0.5 at 1 cm: c = 0.5 / 17,335 = 28.8 µM, or 0.346 mg/mL with the weight; with all cysteines reduced, 29.5 µM.
• The His6 tag adds no absorbance at 280 nm but 2,244 Da of mass, so the mg/mL answer changes.

Method

• Molar extinction coefficient at 280 nm. Pace CN, Vajdos F, Fee L, Grimsley G, Gray T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci 1995;4:2411-2423 (read 5 October 2026).Checked against Biopython 1.85 ProtParam exactly on 22 sequences (6 October 2026); Biopython agrees with recorded ExPASy ProtParam outputs on 9 of them.
• Average molecular weight from the sequence. ExPASy ProtParam (Gasteiger et al. 2005); the fixtures are ProtParam's printed weights, retrieved 5 October 2026.Checked against recorded ExPASy ProtParam outputs within 0.02 Da on 9 UniProt sequences (5 October 2026), the protein app's own fixtures.
• Protein concentration from A280. Beer-Lambert law with the Pace 1995 ε280 (src/lib/seq/protein-mass.ts).Checked against exact rational arithmetic (Python fractions, 3.13.16) on 14 cases, ten of them refusals, with ε280 and the molar mass as inputs; tolerance relative 1e-9, refusals exact; 17 October 2026. ε280 is checked against Biopython 1.85 ProtParam exactly on 22 sequences (6 October 2026).
• Nucleic acid concentration from A260 (rules of thumb). Rules of thumb, not measurements: 50 (dsDNA) and 40 (RNA) µg/mL per A260 unit as printed by Thermo Fisher (read 6 October 2026); 33 for ssDNA, the convention NanoDrop instruments apply (Thermo Fisher NanoDrop One FAQ and technical note MCS-TN1315-EN, read 17 October 2026), from ε260 10 mM⁻¹ cm⁻¹ per nucleotide (Cavaluzzi and Borer 2004, Nucleic Acids Res 32:e13, who measured 38 for non-repetitive ssDNA).Checked against exact rational arithmetic (Python fractions, 3.13.16) on 19 cases, seven of them refusals; tolerance relative 1e-9, refusals exact; 17 October 2026. The factors are conventions transcribed from Thermo Fisher and Cavaluzzi and Borer 2004, not measurements. Dilution is applied before the engine is called.

Constants

• pace-1995: Pace et al. 1995 coefficients: 5,500 Trp, 1,490 Tyr, 125 cystine
• protparam: ExPASy ProtParam average residue masses
• a260-rules: A260 rules of thumb: 50 (dsDNA) and 40 (RNA) as Thermo Fisher prints them; 33 (ssDNA), the NanoDrop convention (Thermo Fisher, read 17 October 2026; Cavaluzzi and Borer 2004 measured 38)

Limits

• For research and education only, and not medical advice: the stock, dilution and buffer calculations here are not for dosing or clinical preparation.
• ε280 is the Pace et al. 1995 estimate for a folded protein in water; the measured value can differ by several percent, and a tag changes it, so the sheet computes the tagged construct when you give it.
• The A260 conversions (50 µg/mL per unit for double-stranded DNA, 40 for RNA, 33 for single-stranded DNA) are rules of thumb; an oligonucleotide's ε260 by nearest neighbour is not computed here.

Verification