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
Introduction
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, so you can move straight from a plate reader or spectrophotometer to a usable number for your protocol.
About the Tool
The Protein Concentration Calculator gives you three independent ways to determine how much protein is in a sample, matched to three common sources of laboratory data. The A280 / Beer-Lambert tab is for a purified protein whose molar extinction coefficient (ε) is known and returns both molar (µM, nM) and mass (mg/mL, µg/mL) concentrations. The E1% tab is for proteins characterised by a manufacturer's or pharmacopoeial specific absorbance value rather than a molar ε, and returns a mass concentration directly. The Standard Curve tab is for colorimetric assays such as BCA or Bradford, where you already have a slope and intercept from a regression of your own standards.
All three tabs share the same dilution-factor field, so if you diluted a sample before reading it, the calculator will back-calculate the concentration of your original, undiluted stock.
Input Explanation
A280 / Beer-Lambert tab: Enter your measured A280 absorbance, the molar extinction coefficient (ε) in M⁻¹cm⁻¹, and the cuvette pathlength in centimetres (default 1 cm). Molecular weight in Daltons is optional and only needed if you want mg/mL and µg/mL alongside µM and nM.
E1% tab: Enter the A280 reading, the E1% value from a data sheet or monograph, and the pathlength. This tab does not need a molecular weight, since E1% is already a mass-based value.
Standard Curve tab: Enter the slope (in absorbance units per µg/mL) and intercept from your BCA or Bradford standard curve regression, along with your sample's measured absorbance.
Dilution factor (all tabs): Leave at 1 if the sample was read undiluted. If you diluted the sample before reading — for example, to bring a strong solution into the linear range — enter that dilution factor so the result reflects the original stock concentration.
Formula Explanation
The Beer-Lambert law underlies 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). Rearranged for concentration: C (mol/L) = A / (ε × l). Multiplying C by the molecular weight (g/mol) converts molar concentration directly to mg/mL, since 1 mol/L × 1 g/mol numerically equals 1 g/L, and 1 g/L is the same value as 1 mg/mL.
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 that unit basis into mg/mL.
For standard curve methods: C (µg/mL) = (A − intercept) / slope, taken directly from the linear regression equation of the standard curve (Abs = slope × C + intercept).
Worked Example
Sample Input
Using the A280 / Beer-Lambert tab: A280 = 0.72, ε = 43,890 M⁻¹cm⁻¹, pathlength l = 1 cm, molecular weight = 45,000 Da, dilution factor = 1 (sample read neat).
Step-by-Step Calculation
1. Apply the rearranged Beer-Lambert formula: C (mol/L) = A / (ε × l) = 0.72 / (43,890 × 1) = 1.6405 × 10⁻⁵ mol/L.
2. Convert to µM and nM: 1.6405 × 10⁻⁵ mol/L × 10⁶ = 16.4046 µM; × 10⁹ = 16,404.65 nM.
3. Convert to mass concentration using the molecular weight: 1.6405 × 10⁻⁵ mol/L × 45,000 g/mol = 0.7382 mg/mL.
4. Convert mg/mL to µg/mL: 0.7382 mg/mL × 1000 = 738.21 µg/mL.
5. No dilution correction is needed here, since the dilution factor is 1.
Final Result
Concentration ≈ 16.40 µM (16,404.65 nM), equivalent to 0.7382 mg/mL (738.21 µg/mL).
Interpretation
A 45 kDa protein at an A280 of 0.72, read undiluted in a 1 cm cuvette with this ε, corresponds to a moderately concentrated stock of roughly 0.74 mg/mL. This is a realistic reading straight off most spectrophotometers without dilution — for comparison, a solution above about A280 = 1.0 would fall outside the linear range and should be diluted and re-read.
Result Interpretation
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 loading, you typically need mg/mL or µg/mL. For enzyme kinetics, receptor binding assays, or other stoichiometric reactions, µM is the preferred unit. A dilution factor greater than 1 means the displayed concentration already reflects the original, undiluted stock rather than the diluted sample you actually read.
Practical Applications
Use the A280 Beer-Lambert tab when working with a purified, well-characterised protein whose extinction coefficient is known or has been calculated from its 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 a pharmacopoeial monograph or with commercial proteins whose data sheets provide E1% 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.
Scientific Notes & Limitations
The Beer-Lambert law is linear only at absorbances below roughly 1.0 AU; above that, light scattering and detector response cause the relationship to break down, and calculated concentrations become unreliable. It also assumes the extinction coefficient you enter accurately reflects your protein's actual tryptophan, tyrosine, and cystine content — a value calculated for the wrong isoform, oxidation state, or a different construct will not be accurate for your sample.
A280 readings can be inflated by anything else in the sample that absorbs near 280 nm, most notably nucleic acids, which absorb strongly at 260 nm but also contribute at 280 nm. An A260/A280 ratio above about 0.6 suggests nucleic acid contamination is affecting the result; the Warburg-Christian correction (C mg/mL = 1.55 × A280 − 0.76 × A260) can partially compensate, but a colorimetric assay such as BCA is generally more robust for impure samples.
None of the three methods in this calculator directly measures protein mass the way a gravimetric or amino acid analysis would — they are all indirect, formula-based estimates, and their accuracy is only as good as the ε, E1%, or standard curve parameters you provide.
Practical Tips
Always blank your spectrophotometer with the same buffer your sample is in — a mismatched blank shifts every subsequent A280 reading. Take at least two or three replicate readings and use the average rather than a single measurement, especially near the edges of the instrument's reliable range.
If you have both A260 and A280 values, check the A260/A280 ratio as a quick contamination check before trusting the A280 concentration. For antibodies or other proteins with a manufacturer-supplied E1%, confirm the value is specific to your exact product and lot, since E1% can vary between formulations of "the same" protein.
If your undiluted reading is above about A280 = 1.0, dilute the sample, re-read it, and enter the dilution factor here rather than trusting a reading taken outside the linear range.
Common Mistakes to Avoid
Wrong pathlength: Microvolume instruments such as the NanoDrop normalise all readings to a 1 cm equivalent, so 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.
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.