Protein Concentration Calculator
Calculate protein concentration using either a standard curve (Bradford/BCA/Lowry) or UV absorbance at 280 nm.
Why this protein concentration calculator matters
If you run gels, enzyme assays, Western blots, or purification workflows, a reliable protein concentration number is non-negotiable. Too low and your assay signal disappears. Too high and you overload columns, membranes, and reaction mixes. This calculator helps you move quickly from raw absorbance values to useful concentration estimates.
Two common ways to calculate protein concentration
1) Standard curve method (Bradford, BCA, Lowry)
Most colorimetric assays generate a linear equation from standards:
y = mC + b
Where:
- y is absorbance of your sample
- m is slope of the standard curve
- b is y-intercept
- C is protein concentration
Rearranging gives:
C = (y - b) / m
If your sample was diluted before reading, multiply by the dilution factor to recover the original concentration.
2) A280 method using extinction coefficient
For purified proteins, you can estimate concentration by UV absorbance:
C (M) = A280 / (ε × l)
Where ε is the molar extinction coefficient and l is path length in cm. If you know molecular weight, convert molarity to mass concentration.
How to use this tool correctly
- Blank your spectrophotometer with the same buffer used for samples.
- Confirm your sample absorbance falls inside the linear range of your standard curve.
- Use the exact dilution factor from your workflow (not a rough guess).
- Keep units consistent with your standard preparation.
- For A280, use an accurate extinction coefficient and molecular weight.
Example calculations
Standard curve example
Suppose your curve is y = 0.80C + 0.02 and your sample absorbance is 0.50, diluted 1:4.
C = (0.50 - 0.02) / 0.80 = 0.60 mg/mL (in diluted sample)
Original concentration: 0.60 × 4 = 2.40 mg/mL
A280 example
If A280 = 0.9, ε = 43,824 M⁻¹ cm⁻¹, l = 1 cm, and MW = 50,000 g/mol:
M = 0.9 / (43824 × 1) = 2.05 × 10⁻⁵ M
mg/mL = M × MW = 1.03 mg/mL
Common pitfalls
- Using a curved region of the assay: Your standards may be linear only in a narrow range.
- Forgetting dilution correction: This is one of the most frequent mistakes in notebooks.
- Mismatched standards: BSA standards may not perfectly match your target protein response.
- Dirty cuvettes or bubbles: Can inflate absorbance and distort concentration.
- Wrong path length: Especially important for microvolume instruments.
FAQ
Can I use this for peptide concentration?
Yes, if your assay chemistry is validated for your peptide and your standard curve is appropriate.
What if the calculator returns a negative value?
This usually means your sample absorbance is near or below the intercept/noise level. Recheck blanking, repeat the assay, or concentrate the sample.
Should I trust A280 or BCA more?
For clean, purified proteins with known extinction coefficients, A280 is fast and reliable. For mixed lysates or unknown compositions, BCA/Bradford with a proper standard curve is often safer.
Final note
This calculator is intended for quick lab calculations and educational use. For publication-quality results, always include replicates, report assay conditions, and verify concentration by orthogonal methods when possible.