Protein Concentration Calculator
Find protein concentration from an A280 absorbance reading using the Beer-Lambert law, or from a Bradford/BCA assay standard curve.
🧪 What is the Protein Concentration Calculator?
The protein concentration calculator finds how much protein, in mg/mL or µg/mL, is present in a sample using two standard laboratory quantification methods: the A280 (Beer-Lambert) method based on direct UV absorbance, and the standard curve method used by Bradford and BCA colorimetric assays. Both are core techniques used constantly in biochemistry, molecular biology, and protein purification workflows.
This calculator is used across many real-world lab contexts. Researchers purifying a recombinant protein use the A280 method for a fast, reagent-free concentration check straight from a NanoDrop or UV spectrophotometer reading. Labs running total-protein assays on cell lysates, tissue extracts, or complex mixtures use the Bradford or BCA standard curve method because it works regardless of a specific protein's amino acid composition. Quality control teams in biomanufacturing use both methods side by side to cross-validate concentration results before formulation or downstream processing.
A common misconception is that these two methods will always agree closely on the same sample. They often do not, A280 measures a specific protein's own UV absorbance (which depends on its tryptophan, tyrosine, and cystine content), while Bradford and BCA measure a colorimetric response to overall protein structure calibrated against a different reference protein (usually BSA), so different proteins can give meaningfully different readings between methods even at identical true concentrations.
This calculator is useful because it handles both formulas correctly, including dilution factors, and shows every step of the arithmetic, so you can quickly verify a concentration reading from either a spectrophotometer or a plate-reader assay without doing the unit conversions by hand.
📐 Formula
📖 How to Use This Calculator
Steps
💡 Example Calculations
Example 1 — A280 with a Generic Extinction Coefficient
A280 = 0.8, ε = 1.0 (mg/mL)-1cm-1, path = 1 cm, dilution = 1
Example 2 — A280 for a Diluted BSA Sample
A280 = 1.5, ε = 0.667 (mg/mL)-1cm-1 (BSA), path = 1 cm, diluted 2×
Example 3 — Standard Curve (BCA Assay) for a Diluted Lysate
Absorbance = 0.55, slope = 0.0015 AU per µg/mL, intercept = 0.05, diluted 10×
❓ Frequently Asked Questions
🔗 Related Calculators
What is the A280 (Beer-Lambert) formula for protein concentration?
Concentration (mg/mL) = (A280 x dilution factor) / (extinction coefficient x path length). The extinction coefficient is specific to each protein and is usually expressed in (mg/mL)^-1cm^-1 for this mass-based form of the Beer-Lambert law.
What extinction coefficient should I use for A280?
Use your specific protein's known extinction coefficient if available, calculated from its tryptophan, tyrosine, and cystine content (many databases and tools such as ExPASy ProtParam provide this). If unknown, 1.0 (mg/mL)^-1cm^-1 is a commonly used generic estimate for an average globular protein, though it can be off by 50% or more for atypical proteins.
What is the standard curve (Bradford/BCA) formula?
Concentration (µg/mL) = [(measured absorbance - intercept) / slope] x dilution factor, where slope and intercept come from a linear regression fit to a set of known-concentration protein standards (commonly BSA) measured on the same assay.
Should I use the A280 method or a Bradford/BCA assay?
A280 is fast, non-destructive, and needs no reagents, but requires a known or estimated extinction coefficient and only works if the protein absorbs at 280 nm. Bradford and BCA assays work for essentially any protein and give a colorimetric readout independent of amino acid composition, but require reagents, a standard curve, and destroy the sample (or an aliquot of it).
Why is my Bradford/BCA result different for two different proteins at the same true concentration?
Both Bradford and BCA assays respond to a protein's amino acid composition (Bradford binds preferentially to arginine and aromatic residues, BCA responds to peptide bonds and certain residues), so two proteins at identical true concentrations can give different absorbance readings against a BSA standard curve, this is a well-known source of assay-to-assay variability.
What is a typical BCA or Bradford assay slope and intercept?
Values vary by kit, plate reader, and protein standard, but a linear BCA standard curve often has a slope in the range of roughly 0.001 to 0.003 absorbance units per µg/mL and a small positive intercept (the blank reading), always generate your own standard curve rather than reusing a fixed slope and intercept from a different assay run.
How does dilution factor work in this calculator?
Enter the dilution factor as the multiplier that converts your diluted sample's measured absorbance back to the concentration of your original, undiluted sample. For example, if you diluted a sample 1:10 before measuring, enter a dilution factor of 10, and the calculator multiplies the computed concentration by 10.
Why does the calculator show an error when absorbance is below the standard curve intercept?
In standard curve mode, if your measured absorbance is lower than the intercept (blank reading), the formula would produce a negative concentration, which is not physically meaningful. This usually indicates measurement noise near zero concentration, an incorrect blank, or a sample that truly has negligible protein.
Can nucleic acid contamination affect A280 protein readings?
Yes. DNA and RNA absorb strongly near 260 nm but also contribute meaningfully at 280 nm, so a nucleic-acid-contaminated sample will read a falsely high A280 protein concentration. Checking the A260/A280 ratio (roughly 0.57 for pure protein, much higher for nucleic acids) helps flag this issue before trusting an A280-based concentration.
What path length should I use?
Path length should match your actual cuvette or plate well, standard cuvettes are 1 cm, but microplate readers often use shorter effective path lengths (commonly around 0.5 cm for a typical 200 microliter well), check your instrument's specification or use its built-in path-length correction if available.
Does this calculator work for antibody or IgG concentration?
Yes, the same A280 Beer-Lambert formula applies to antibodies, which commonly use an extinction coefficient around 1.4 (mg/mL)^-1cm^-1 for IgG, though the exact value varies by antibody isotype and should be confirmed from the specific antibody's datasheet when precision matters.