What is the A280 method for measuring protein concentration?

The A280 method uses the absorbance of aromatic amino acids (tryptophan and tyrosine) at 280 nm. The concentration is calculated using the Beer-Lambert law: c = A280 / (epsilon x path length), where epsilon is the molar extinction coefficient of the protein. This method is non-destructive and fast but requires a known extinction coefficient for accurate results.

What is the Bradford assay and when should I use it?

The Bradford assay is a colorimetric method that uses Coomassie Brilliant Blue G-250 dye, which shifts absorption from 465 nm to 595 nm when it binds protein. It is sensitive, fast, and compatible with most buffer conditions. Use Bradford when your sample contains nucleic acid contamination (which interferes with A280) or when you need a low-cost, high-throughput method.

What is the BCA assay?

The BCA (bicinchoninic acid) assay is a colorimetric method where cupric ions (Cu2+) are reduced to cuprous ions (Cu+) by protein peptide bonds under alkaline conditions. The cuprous ions then react with BCA reagent to form a purple complex detected at 562 nm. BCA is more sensitive than Bradford and is compatible with detergents, making it useful for membrane proteins.

Why does dilution factor matter in protein quantification?

The spectrophotometer measures the absorbance of the diluted sample, not the original. Multiplying by the dilution factor corrects the reading back to the original sample concentration. For example, if you diluted 1:10 and measured A280 = 0.12, the true concentration reflects a 10-fold correction. Skipping this step produces a 10-fold underestimate.

What extinction coefficient should I use for BSA?

Bovine serum albumin (BSA) has a molar extinction coefficient of approximately 43,824 M-1 cm-1 at 280 nm and a molecular weight of 66,433 Da. These values give a concentration of approximately 0.667 mg/mL per absorbance unit at A280. BSA is the most common protein standard for Bradford and BCA assays.

Can I use the Bradford assay with detergents?

Most ionic and non-ionic detergents interfere with the Bradford assay by competing with the dye or by causing precipitation. SDS at concentrations above 0.1% is particularly disruptive. If your sample contains detergent, use the BCA assay instead, which is compatible with up to 5% SDS, 5% Triton X-100, and other common detergents.

What is the linear range of each protein assay?

The standard Bradford assay is linear from approximately 1 to 1,500 micrograms per mL. The BCA assay is linear from 20 to 2,000 micrograms per mL, and a micro-BCA version extends sensitivity down to 0.5 micrograms per mL. A280 direct measurement is most accurate between absorbance values of 0.1 and 1.0. Samples outside the linear range should be diluted and re-measured.

Why do different proteins give different Bradford readings at the same concentration?

The Bradford dye binds preferentially to arginine and aromatic residues. Proteins with different amino acid compositions bind different amounts of dye, causing variation in A595 for the same mass concentration. This is why the standard curve is typically built with BSA, and results are reported as BSA equivalents. For absolute quantification, use a standard of the same protein or the A280 method with the protein's own extinction coefficient.

What is the difference between molar concentration and mass concentration?

Mass concentration (mg/mL or ug/mL) reports the total mass of protein per unit volume. Molar concentration (uM or nM) reports the number of molecules per unit volume. Molar concentration is calculated by dividing mass concentration by the molecular weight. For enzymatic assays or binding studies where stoichiometry matters, molar concentration is more informative.

How do I calculate the extinction coefficient for my own protein?

The molar extinction coefficient at 280 nm can be predicted from the amino acid sequence using the ProtParam tool on ExPASy. The calculation counts tryptophan (5,500 M-1 cm-1), tyrosine (1,490 M-1 cm-1), and cystine disulfide bonds (125 M-1 cm-1 per bond). The predicted value is accurate to within 5 to 10% for most folded proteins.

Protein Concentration Calculator - A280, Bradford, BCA

Protein Concentration Calculator

The Protein Concentration Calculator determines protein concentration from absorbance readings using three methods: direct A280 measurement with the Beer-Lambert law, Bradford assay at A595, and BCA assay at A562. Select your method, enter the absorbance and dilution factor, and the calculator returns concentration in micrograms per milliliter with the applied formula.

What Is the Protein Concentration Calculator?

The Protein Concentration Calculator determines how much protein is present in a sample based on absorbance readings from a spectrophotometer. It supports three standard laboratory methods: A280 direct measurement using the Beer-Lambert law, the Bradford assay at 595 nm, and the BCA assay at 562 nm. Each method suits different experimental conditions, sample types, and accuracy requirements.

Accurate protein quantification is a prerequisite for downstream applications including gel electrophoresis, Western blotting, enzyme activity assays, and structural studies. This calculator eliminates manual arithmetic and presents results in micrograms per milliliter alongside the formula applied.

The Formulas Behind Each Method

All three methods use a form of the Beer-Lambert law, which states that absorbance is proportional to concentration:

\[ c = \frac{A}{\varepsilon \times l} \]

Where A is the measured absorbance, epsilon is the extinction coefficient, and l is the path length in centimeters (1 cm for standard cuvettes). For colorimetric assays, empirical conversion factors replace the extinction coefficient:

A280 Direct: c (mg/mL) = A280 / epsilon_280, where epsilon_280 is the protein's known molar extinction coefficient divided by its molecular weight
Bradford (A595): c (ug/mL) = (A595 - 0.001) / 0.0012 x dilution factor
BCA (A562): c (ug/mL) = (A562 - 0.04) / 0.0003 x dilution factor

A280 Direct Method: Protein-Specific Accuracy

The A280 method is non-destructive and requires no reagents beyond the spectrophotometer. Its accuracy depends on knowing the extinction coefficient of the specific protein being measured. Tryptophan residues contribute 5,500 M-1 cm-1, tyrosine residues contribute 1,490 M-1 cm-1, and cystine disulfide bonds contribute 125 M-1 cm-1 each. For BSA, the molar extinction coefficient is 43,824 M-1 cm-1 at 280 nm with a molecular weight of 66,433 Da.

A limitation of A280 is that nucleic acids also absorb at 280 nm. Samples contaminated with DNA or RNA will overestimate protein concentration. The A260/A280 ratio can indicate nucleic acid interference: a ratio above 0.6 suggests nucleic acid contamination requiring cleanup before A280 measurement.

Bradford vs. BCA: Choosing the Right Colorimetric Assay

When the protein's extinction coefficient is unknown or when nucleic acid interference is likely, colorimetric assays offer an alternative. The two most common are Bradford and BCA:

Bradford assay: Fast (10 minute incubation), room temperature, sensitive from 1 to 1,500 ug/mL. Incompatible with detergents above 0.1% SDS or with reducing agents at high concentration.
BCA assay: Sensitive from 20 to 2,000 ug/mL, compatible with up to 5% SDS and most non-ionic detergents. Requires 30-60 minute incubation at 37 degrees C. Results can be affected by reducing agents like DTT.

For routine protein quantification without detergents, Bradford is faster. For membrane proteins or samples containing SDS from a previous purification step, BCA is the better choice.

Accuracy and Limitations

All three methods assume the sample is within the linear range of the assay. For A280, absorbance values between 0.1 and 1.0 give the most reliable results. Values above 1.0 may show stray light artifacts depending on the spectrophotometer. For Bradford and BCA, results are reported as equivalents of the standard protein (usually BSA), meaning that a glycine-rich protein and a tryptophan-rich protein at the same mass concentration may read differently. The dilution factor correction is critical: a tenfold dilution that is not corrected produces a tenfold underestimate of the original concentration.

Real-World Case Study: Cambridge, UK, March 2022

In March 2022, a biochemistry PhD student at the University of Cambridge was purifying recombinant GFP from an E. coli expression system. After the final size exclusion chromatography step, she collected 2 mL of eluate. Using A280 measurement with the known GFP extinction coefficient of 55,000 M-1 cm-1 and molecular weight of 27,000 Da, she obtained A280 = 0.38 (dilution factor 1, path length 1 cm). The calculator returned 0.186 mg/mL, giving a total of 0.372 mg GFP in the 2 mL fraction. She verified this with a Bradford assay against a BSA standard curve, which returned 0.19 mg/mL, confirming agreement within 2%. This cross-validation confirmed the purification yield before proceeding to crystallography experiments.

Expert Insight from Muhammad Shahbaz Siddiqui, Founder, TheCalculatorsHub

In building and researching the tools on this site, the most common mistake I see is failing to check whether the sample is within the assay's linear range before reporting a concentration. A Bradford reading of 1.8 absorbance units is outside the linear range for standard Coomassie and will underestimate concentration significantly. Always dilute and remeasure if the reading exceeds the standard curve. My second recommendation is to always run a standard curve on the same day as your samples, as room temperature and reagent lot variation can shift the curve by 5 to 15% between sessions.

Applying Protein Quantification in Research

Protein concentration data feeds directly into experimental planning. For Western blotting, a standard loading of 10 to 50 ug per lane requires knowing the exact concentration to load equal amounts across samples. For enzyme kinetics assays, molar concentration is needed to calculate specific activity (units per mg) and compare catalytic efficiency across conditions. In structural biology, sample concentration must reach 5 to 20 mg/mL for crystallization trials, and the A280 method is used routinely to monitor concentration during concentration steps using ultrafiltration devices.

Protein Concentration Calculator