This free protein concentration calculator supports three quantification methods — Beer-Lambert (A280), the E1% specific absorbance method, and standard curve interpolation for BCA or Bradford assays. Used by biochemists, protein purification scientists, and structural biologists, it instantly converts spectrophotometric readings into concentration values in µM, nM, mg/mL, and µg/mL.
Protein Concentration Calculator
FREE TOOLUse our Extinction Coefficient Calculator above.
Required for mg/mL output.
If sample was diluted before reading.
E1% is the absorbance of a 1% (10 mg/mL) protein solution at 1 cm pathlength. It can be found in protein data sheets or calculated from ε and MW.
A280 of 10 mg/mL at 1 cm.
Enter the slope and intercept from a BCA or Bradford standard curve (Absorbance = slope × concentration + intercept).
💧 Protein Concentration Results
Calculation Steps
How to Use the Protein Concentration Calculator
This calculator provides three independent methods for determining protein concentration from spectrophotometric measurements. Selecting the correct method depends on what information you have available about your protein and sample purity.
Step-by-Step Instructions
A280 / Beer-Lambert tab: Enter your measured A280 absorbance value, the molar extinction coefficient (ε) in M⁻¹cm⁻¹, and the cuvette pathlength in centimetres (default 1 cm). Optionally enter the molecular weight in Daltons to receive output in mg/mL alongside µM. If your sample was diluted before reading, enter the dilution factor to back-calculate the original stock concentration.
E1% tab: If you have the specific absorbance value (E1%) from a manufacturer data sheet or pharmacopoeial monograph, enter the A280 reading, E1%, and pathlength. This method is particularly common for antibodies and albumin where E1% values are well-established, and it does not require knowledge of the molecular weight.
Standard Curve tab: For BCA or Bradford assays, run your standards, plot absorbance vs. concentration, and fit a linear regression. Enter the slope (AU per µg/mL) and intercept from that regression along with the sample absorbance. The calculator will interpolate the concentration and apply any dilution correction.
The Scientific Formula Used
The Beer-Lambert law is the foundation of direct A280 measurements: A = ε × C × l, where A is absorbance (dimensionless), ε is the molar extinction coefficient (M⁻¹cm⁻¹), C is molar concentration (mol/L), and l is pathlength (cm). Rearranging for concentration gives C (mol/L) = A / (ε × l). To convert to mg/mL, multiply by molecular weight (g/mol) and divide by 1000.
The E1% formula is: C (mg/mL) = A280 × 10 / (E1% × l). E1% is defined as the absorbance of a 10 mg/mL protein solution at 1 cm pathlength, so multiplying by 10 converts the unit basis.
For standard curve methods: C (µg/mL) = (A − intercept) / slope, derived directly from the linear regression equation of the standard curve.
When to Use This Calculator
Use the A280 Beer-Lambert tab when working with a purified, well-characterised protein whose extinction coefficient is known or has been calculated from sequence (e.g. via ExPASy ProtParam). This is the fastest, most precise method and is non-destructive — no reagents are consumed. It is standard practice in structural biology, enzymology, and antibody characterisation workflows.
Use the E1% method when working with proteins listed in the European Pharmacopoeia or with commercial proteins that provide E1% values instead of ε. It is also useful when molecular weight is uncertain or when preparing high-concentration formulations where mass concentration matters more than molar concentration.
Use the Standard Curve tab when your sample is impure, is a crude cell lysate, or contains interfering substances that would compromise direct A280 measurements. BCA and Bradford assays generate reliable results across a wide dynamic range and are the standard for total protein quantification in cell biology, proteomics, and biochemical fractionation workflows.
Common Mistakes to Avoid
Wrong pathlength: Microvolume instruments such as the NanoDrop normalise all readings to 1 cm equivalent, so always leave pathlength at 1 cm when entering NanoDrop values. Non-standard cuvettes or plate reader wells require you to measure and enter the actual pathlength.
Ignoring nucleic acid contamination: If your A260/A280 ratio exceeds 0.6, nucleic acids are co-purifying with your protein. A280 will systematically overestimate protein concentration; switch to BCA or apply the Warburg-Christian correction (C mg/mL = 1.55 × A280 − 0.76 × A260).
Forgetting the dilution factor: If you diluted your sample 10-fold before reading to bring absorbance into the linear range, you must enter 10 in the dilution factor field. Without this correction, the reported concentration will be 10-fold too low.
Using the wrong extinction coefficient: Extinction coefficients are protein-specific and sequence-dependent. Using a generic or literature value from a different protein isoform can lead to significant errors. Always compute ε from your actual sequence, or use the Extinction Coefficient Calculator on this site.
Exceeding the linear range: The Beer-Lambert law is linear only at absorbances below about 1.0 AU. At higher readings, the relationship breaks down and concentration will be underestimated. Dilute your sample and re-read if A280 exceeds 1.0.
Interpreting Your Results
The calculator reports concentration in µM and nM (molar units, requiring ε as input) and in mg/mL and µg/mL (mass units, requiring both ε and molecular weight, or using E1%). For downstream applications such as SDS-PAGE, you typically need mg/mL or µg/mL. For enzyme kinetics, receptor binding assays, or stoichiometric reactions, µM is the preferred unit. A dilution factor > 1 means the displayed concentration reflects the original undiluted stock.
Frequently Asked Questions
What extinction coefficient should I use for my protein at A280?
The molar extinction coefficient (ε) at 280 nm depends on the amino acid composition of your protein, specifically the content of tryptophan, tyrosine, and cystine residues. For most proteins, ε can be predicted from the sequence using tools such as ExPASy ProtParam. Typical values range from around 1,000 M⁻¹cm⁻¹ for small peptides to over 200,000 M⁻¹cm⁻¹ for large multi-domain proteins. If your protein lacks tryptophan and tyrosine, the A280 method is unreliable and you should use BCA or Bradford assays instead. You can also use our Extinction Coefficient Calculator to compute ε directly from your protein sequence.
How do I account for nucleic acid contamination in A280 measurements?
Nucleic acids absorb strongly at 260 nm and also contribute to absorbance at 280 nm, which causes the Beer-Lambert method to overestimate protein concentration. A common correction is the Warburg-Christian formula: Protein (mg/mL) = 1.55 × A280 − 0.76 × A260. If your A260/A280 ratio is above 0.6, nucleic acid contamination is likely significant. In such cases, it is best to either purify the protein further, treat with DNase/RNase, or switch to a colorimetric assay like BCA, which is less affected by nucleic acids.
What is E1% and when should I use it instead of the molar extinction coefficient?
E1% (also written as E¹%₁cm) is the absorbance of a 1% (w/v) protein solution — equivalent to 10 mg/mL — measured at 1 cm pathlength. It is a mass-based specific absorbance value commonly listed in pharmacopeial monographs and manufacturer protein data sheets for antibodies, albumin, enzymes, and other well-characterised proteins. The E1% method is useful when you do not know the exact molecular weight of your protein or when working with heterogeneous protein preparations such as polyclonal antibody mixtures. The formula is C (mg/mL) = A280 × 10 / (E1% × l).
When should I use a BCA or Bradford standard curve instead of direct A280?
Direct A280 is ideal for pure proteins with a known extinction coefficient, but it becomes unreliable in crude lysates, heterogeneous mixtures, or samples containing nucleic acids, coloured cofactors, or UV-absorbing buffers. BCA (bicinchoninic acid) assays work well across a wide concentration range of roughly 1 to 2000 µg/mL, are compatible with most detergents, and are insensitive to nucleic acid contamination. Bradford assays are faster and cheaper, but are incompatible with detergents such as SDS or Triton X-100 and show protein-to-protein variability. Use the Standard Curve tab in this calculator by entering the slope and intercept values derived from your standard curve regression.
How does the pathlength affect protein concentration calculations?
According to the Beer-Lambert law, absorbance is directly proportional to pathlength: A = ε × C × l, where l is the pathlength in centimetres. Standard cuvettes have a 1 cm pathlength, but microvolume spectrophotometers such as the NanoDrop use shorter pathlengths (0.2 mm or 1 mm) and automatically normalise readings to a 1 cm equivalent. If you are using a non-standard cuvette or a plate reader, you must enter the actual pathlength so the calculator can apply the correct denominator. Using an incorrect pathlength is one of the most common sources of concentration error in protein quantification.