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Bradford Protein Assay Calculator

Calculate protein concentrations from a Bradford (Coomassie) assay: fit the standard curve as a line or a curve, read the unknowns off it and apply dilution factors.

Formula

A595=ac2+bc+d(or A595=bc+d)A_{595} = a c^2 + b c + d\quad(\text{or } A_{595} = b c + d)
c=root of the fitted curve for the measured Ac = \text{root of the fitted curve for the measured } A
coriginal=c×DFc_{\text{original}} = c \times \mathrm{DF}
cc
protein concentration in the well, in the units of the standards
a, b, da,\ b,\ d
coefficients of the fitted curve

How it works

In the Bradford assay, Coomassie Brilliant Blue shifts its absorbance maximum from 465 to 595 nm on binding protein, mostly through arginine and, to a lesser extent, the aromatic residues. The colour is read at 595 nm and compared with a standard curve of a protein of known concentration, usually BSA.

The response is not linear over a wide range, so the tool offers a straight line and a second-order polynomial, and shows the residuals so you can judge which suits your standards. The calibration model is explicit: the equation, R², the residual SD and the plot are displayed, and any unknown outside the standards is flagged rather than extrapolated silently.

Worked example

Eight BSA standards from 0 to 2,000 µg/mL (illustrative absorbance values) and two lysates diluted 10- and 5-fold.

  1. The second-order fit is A = −1.193 × 10⁻⁷ c² + 6.381 × 10⁻⁴ c + 0.0076, R² = 0.9997.
  2. Lysate A (A = 0.240) gives 393.1 µg/mL in the well, times 10 = 3,931 µg/mL.

Lysate A is about 3,930 µg/mL and lysate B 3,330 µg/mL (0.380 gives 666.7 in the well, times 5).

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • The unknown proteins respond to the dye like the protein used for the standards, which is often not quite true.
  • The unknowns and standards are in the same buffer, read at the same time.
  • The standards' absorbances here are illustrative; use your own measured values.

Common mistakes

  • Using a straight line over a range where the assay curves, which under- or over-reads at the ends.
  • Reading an unknown whose absorbance lies beyond the top standard instead of diluting it.
  • Forgetting that detergents and strongly basic buffers interfere, and that Bradford values are relative.

Related tools

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Related equipment

Service documentation, failure modes and parts for the instruments this calculation is used with.