Protein Concentration Calculator (Bradford · BCA · A280)

How calculations work

• Bradford/BCA: Use a standard curve A = m·C + b. Concentration: C = (A_sample − b)/m. You may paste standards to fit m and b (least squares), with optional zero-intercept.
• A280: Beer–Lambert law, A = ε·l·c. • If using molar ε: c (mg/mL) = (A·MW) / (ε·l). • If using mass ε: c (mg/mL) = A / (ε·l).
• Dilution factor multiplies the calculated concentration.

Example protein concentration calculations

Example 1: Bradford concentration from a standard curve

A Bradford standard curve gives A = 0.470C + 0.002, where concentration is in mg/mL. An unknown sample has absorbance replicates of 0.235, 0.236, and 0.233. The mean absorbance is 0.2347, so the blank-adjusted concentration is (0.2347 - 0.002) / 0.470 = 0.495 mg/mL. If the sample was diluted 1:10 before reading, report 4.95 mg/mL.

Example 2: BCA concentration with dilution factor

A BCA sample reads 0.620 after blank subtraction. The fitted curve gives 0.310 mg/mL for the assay well. If the original sample was diluted 1:5, multiply by 5: 0.310 × 5 = 1.55 mg/mL. When BCA standards show visible curvature, use the quadratic fit only if the standards bracket the unknown and residuals improve.

Example 3: A280 concentration using Beer-Lambert law

A protein has A280 = 0.850, molecular weight 66,000 Da, molar epsilon 55,000 M^-1cm^-1, and path length 1 cm. The concentration is (0.850 × 66000) / (55000 × 1) = 1.02 mg/mL. If reporting molarity, divide g/L by molecular weight: 1.02 / 66000 = 15.5 μM.

Example 4: Out-of-range sample and what to do

If standards span 0.0 to 1.0 mg/mL but the unknown calculates to 1.8 mg/mL, the result is an extrapolation. Dilute the sample, rerun the assay, and choose a dilution that places the unknown near the middle of the standard curve. If the unknown is below the lowest standard, use more sample, concentrate it, or prepare lower standards.

How to prepare a protein standard curve

Recommended BSA standard ranges for BCA and Bradford

A typical Bradford curve often covers about 0.1-1.0 mg/mL for cuvette-style readings, while many BCA protocols cover a broader range such as 0.02-2.0 mg/mL depending on reagent format and incubation. Use the range specified by your kit or validated lab protocol, and make sure unknown samples fall inside the standard range after dilution.

How many standards should I use?

Use at least five non-zero standards plus a blank for routine work. Seven to eight standards give a better view of curvature and make residuals easier to interpret. Run standards and unknowns in duplicate or triplicate when sample volume allows.

How to handle blanks and buffer-matched standards

The blank should contain the same buffer and assay reagent as the standards, but no protein. If your unknowns contain detergents, reducers, salts, imidazole, glycerol, or other additives, prepare buffer-matched standards whenever possible so the matrix affects standards and unknowns similarly.

How to interpret R², RMSE, residuals, and outliers

R² shows how much variation is explained by the fitted curve, but it does not prove the fit is appropriate. RMSE gives the typical absorbance error. Residuals should be small and randomly scattered; a curved residual pattern means a linear model may be inappropriate or the standards may exceed the assay’s linear range. Investigate obvious outliers before excluding them.

Understanding Bradford, BCA, and A280

Protein concentration can be estimated in three common ways: colorimetric dye-binding assays (Bradford and BCA) and direct UV absorbance at 280 nm (A280). While all three aim to report the same thing—how much protein is present—they differ in chemistry, dynamic range, sensitivity to reagents, and what assumptions they make. This section explains how each approach works, why a standard curve or extinction coefficient is needed, and how to interpret your results with good laboratory practice.

Lowry, Biuret, and custom absorbance assays

Lowry, Biuret, and many plate-reader protein assays use the same standard-curve calculation pattern: measure known standards, fit absorbance versus concentration, then invert the fit for unknowns. Select the matching method or Custom standard curve, enter the assay wavelength, and paste standards in the unit used by your protocol.

Bradford & BCA assays

Bradford and BCA are colorimetric assays that convert protein amount into a measurable absorbance. In practice you measure a series of standards (e.g., BSA) and fit a line of the form A = m·C + b, where A is absorbance and C is concentration in mg/mL (or μg/mL). Your unknown is then computed as C = (A_sample − b)/m. In the linear range these assays behave well, but at high concentrations the curve can deviate from linearity; some labs use a quadratic fit for BCA in the upper range. Always include a blank and several standards spaced across the range that covers your samples.

Matrix effects matter. Detergents, chaotropes, and reducing agents can interact with either dye chemistry (e.g., Bradford is more sensitive to detergents; BCA can be affected by strong reducers unless “compatible” protocols are used). If your sample buffer differs from the standard buffer, consider preparing matrix-matched standards (same buffer composition) or diluting samples and standards into a common diluent. Inspect fit quality using R² and residuals; a non-zero intercept can flag background or plate artifacts. If you force the fit through the origin (common when blanks are excellent), verify that residuals stay random and small.

Direct A280 (Beer–Lambert)

Many proteins absorb strongly at 280 nm due to tryptophan and tyrosine residues. The Beer–Lambert law relates absorbance to concentration via A = ε·l·c, with path length l (cm) and extinction coefficient ε. If you know the molar ε (M⁻¹·cm⁻¹) and the molecular weight, you can compute mass concentration as c(mg/mL) = (A·MW)/(ε·l). If you instead have a mass ε (mL·mg⁻¹·cm⁻¹), use c(mg/mL) = A/(ε·l). Microvolume spectrophotometers may auto-correct path length; confirm whether your instrument reports path-corrected A280 or raw absorbance.

A280 assumes your protein’s ε is known and accurate for the exact sequence and redox state (e.g., disulfide formation slightly changes ε). Nucleic acids, heme, or scattering from particulates can inflate A280; if contamination is likely, consider parallel A260/A280 checks or a dye-based assay as an orthogonal method. For best accuracy, measure within 0.1–1.0 absorbance units and apply the correct dilution factor explicitly.

Good reporting practices

• State the method (Bradford, BCA, or A280), wavelength, path length, and dilution factor.
• For assays, report slope, intercept, number of standards, and fit model (linear vs. forced-zero).
• For A280, report ε (molar or mass) and the molecular weight if applicable.
• Flag out-of-range readings and repeat within the validated dynamic range.
• When precision matters, verify with a second method or an orthogonal standard.

Tip: This calculator lets you paste standards to auto-fit the curve, toggle a zero-intercept, and switch between molar or mass extinction coefficients for A280, keeping units and dilution explicit for transparency.

Protein Concentration Calculator FAQ

How do I calculate protein concentration from absorbance?

Subtract the blank, fit absorbance against standards of known concentration, and solve the fitted equation for the unknown. Multiply by the dilution factor to report the original sample concentration.

What is the formula for Bradford or BCA concentration?

With a linear curve A = mC + b, use C = (A_sample - b) / m. With a forced-zero fit, b = 0. With a quadratic fit, the calculator solves the fitted quadratic and selects the root that best matches the standard range.

Should I subtract the blank from standards and samples?

Yes. Apply blank subtraction consistently to standards and unknowns. This removes absorbance from buffer, reagent, plate, or cuvette background before fitting or calculating concentration.

What dilution factor should I enter?

Enter the total dilution before measurement. A 1:10 dilution uses a dilution factor of 10. Serial dilutions multiply together, so two 1:5 steps use a total factor of 25.

When should I use a linear vs quadratic standard curve?

Use a linear curve when the standards fall in the assay’s linear range and residuals are random. Use a quadratic curve for BCA only when your standards clearly support curvature and the unknown remains within the fitted standard range.

Why is my unknown outside the standard curve range?

The unknown absorbance is above or below the standards used to define the curve. Dilute high samples, concentrate low samples, or rebuild the standard curve so the unknown is bracketed by standards.

How do I calculate protein concentration from A280?

Use Beer-Lambert law. With molar epsilon and molecular weight, mg/mL = A280 × MW / (epsilon × path length). With a mass extinction coefficient, mg/mL = A280 / (epsilon × path length).

What is the difference between Bradford, BCA, and A280?

Bradford and BCA use dye chemistry and require standards. A280 is direct UV absorbance and requires an extinction coefficient. Bradford is quick and sensitive, BCA is often more tolerant of detergents, and A280 is fastest when the protein sequence or extinction coefficient is known.