Protein A280 Concentration by Beer Lambert Calculation
Convert a declared A280 reading to protein concentration using a sequence-derived extinction coefficient and an explicit optical path length. The reference opens the existing GFP protein sequence with illustrative absorbance. It keeps the cysteine convention and molecular-weight basis visible so that mass concentration and molar concentration can be interpreted consistently.
The existing UniProt P42212 reference sequence is paired with A280 = 0.5 and a 1 cm path length as calculation inputs. The absorbance is hypothetical. The reduced-cysteine convention is selected explicitly; this is not a measured concentration of a GFP sample.
State what the optical measurement represents
Absorbance is a measurement relative to a blank under defined optical conditions. The protein concentration equation assumes the measured contribution is attributable to the protein described by the extinction coefficient. Buffer mismatch, contaminants and scattering can violate that assumption. Keep the blank, wavelength and instrument reporting convention with the result instead of retaining only the final concentration.
The coefficient is molar, so the direct output is an amount concentration. Molecular weight then converts that result to mass concentration. This two-step relationship explains why an incorrect construct mass can change mg/mL even when the molar estimate is unchanged. Conversely, an incorrect extinction coefficient affects both outputs because it changes the inferred amount concentration.
Use the sequence estimate within its scope
The existing method counts the relevant residues under a stated cysteine convention. A sequence without the absorbing groups required by the estimate may not support a useful result. The software should preserve that refusal rather than divide by a missing coefficient. The illustrative GFP input is convenient for exploring the workflow, but its actual optical properties can include effects beyond the simple sequence estimate.
For a protein preparation used in quantitative experiments, consider an independent concentration measurement or a measured extinction coefficient when appropriate. Retain the sequence accession or construct sequence, terminal tags, cleavage state and any assumed modifications. Those details make a calculation reproducible and help explain differences between two laboratories that report concentration for nominally the same protein.
Equations and variables
Beer–Lambert relationship
A280 = ε280 × c × l; c = A280/(ε280 × l)
- A280
- Blank-corrected absorbance at 280 nm
- ε280
- Molar extinction coefficient on the stated sequence/convention (L mol⁻¹ cm⁻¹)
- c
- Protein amount concentration (mol/L)
- l
- Optical path length (cm)
Mass concentration
cmass = c × MW
- cmass
- Protein mass concentration (g/L; numerically equivalent to mg/mL)
- MW
- Molar mass of the represented protein (g/mol)
Scope and limitations
- Sequence-based extinction estimates depend on aromatic residues and the stated cysteine convention. Tags, cleavage, modifications and chromophore maturation can affect applicability.
- Nucleic acids, other absorbing molecules, turbidity and scattering can contribute to the reading. The equation does not identify or remove them.
- The optical path must be known or correctly corrected by the instrument. A microvolume reading must not be treated as a raw 1 cm reading without checking its convention.
Sources and provenance
- Calculator inputs and independent arithmetic verificationOriginal synthetic inputs; see individual fixture notices
- UniProtKB GFP P42212CC BY 4.0
- Pace et al.: how to measure and predict a protein molar absorption coefficientCited method; no article data reproduced
Technical questions
Why must the tag and cleavage state match the sequence?
Added or removed residues change molecular weight and can change the predicted extinction coefficient. A sequence for the native protein is not always the sequence of the purified construct. Use the actual construct basis and record any cleavage or modification assumptions.
Can I directly compare a microvolume instrument reading with this example?
Check whether the instrument reports absorbance normalised to a 1 cm path or a reading at its physical path length. Enter a consistent pair of absorbance and path length. Applying a path correction twice can produce a large but plausible-looking concentration error.
Does the GFP example account for every chromophore effect?
No. It illustrates the existing sequence-based calculation. GFP maturation and chromophore-specific absorbance require additional consideration for a real sample. A reference sequence and hypothetical reading do not establish the optical behaviour or purity of your preparation.
Connected workflows
- Six-frame translation and ORF inspection
Inspect the sequence and translation behind a protein construct.
- EGFR sequence and structure inspection
Retrieve a protein sequence with explicit accession and residue numbering.
- 4PL standard-curve interpolation
Compare a direct optical relation with standard-curve interpolation.
- Tris, HEPES and Phosphate Buffer pKa Temperature Shift Tool
Check how a buffer pH set at 25 C shifts in the cold room before a measurement.
- DNA and RNA A260 Concentration and Yield Calculator
Convert an A260 reading to nucleic-acid concentration, the DNA counterpart of A280.