Drybench Assay.
Bradford and BCA protein assay standard curve
Protein concentration from a Bradford or BCA standard curve, as a BSA-equivalent value, with the blank subtracted and nothing read outside the standards.
Bradford and BCA concentrations come from a standard curve run with the samples. The answer is a standard-equivalent concentration: BSA-equivalent when the standards are BSA, because the dye and copper responses differ between proteins. The zero-protein standard is the blank, subtracted before fitting.
Open Standard curve in the appIn the app, choose Standard curve in the Analysis menu, paste your readings, and pick the kit or protocol.
Worked example: a Bradford-style series
Self-generated readings, not kit or manufacturer data, fitted when this page is built by the same engine the app uses and checked against exact rational least squares on the published fixture.
Linear, fitted intercept; tested standard range 125 to 1,000 µg/mL.
| Sample | Mean response | Interpolated (µg/mL) | Original sample (µg/mL) | Status |
|---|---|---|---|---|
| lysate A | 0.392 | 389.2 | 389.2 | Interpolated |
| lysate B | 0.608 | 624.6 | 3,123 | Interpolated |
| too dilute | 0.09 | – | – | Below tested range |
| too concentrated | 1.204 | – | – | Above tested range |
Bradford (Coomassie dye binding): the result is a standard-equivalent concentration, BSA-equivalent when the standards are BSA, because dye binding differs between proteins (Bradford 1976, Anal Biochem 72:248–254). The response is not linear over a wide range: one kit's manual gives 100 to 1,500 µg/mL (standard protocol) and 1 to 25 µg/mL (micro protocol) and prefers a four-parameter or point-to-point curve to a linear fit (Thermo Scientific Pierce Bradford Protein Assay Kit, MAN0011181 Rev. C.0, read 10 October 2026). The zero-protein standard is the blank: assign it as a blank and subtract it.
Worked example: a BCA-style series with its blank subtracted
Self-generated readings, not kit or manufacturer data, fitted when this page is built by the same engine the app uses and checked against exact rational least squares on the published fixture.
Linear, fitted intercept; tested standard range 25 to 2,000 µg/mL.
| Sample | Mean response | Interpolated (µg/mL) | Original sample (µg/mL) | Status |
|---|---|---|---|---|
| sample 1 | 0.308 | 332.6 | 332.6 | Interpolated |
| sample 2 | 0.602 | 658.3 | 6,583 | Interpolated |
| below the lowest standard | 0.012 | – | – | Below tested range |
BCA (bicinchoninic acid): the result is a standard-equivalent concentration, BSA-equivalent when the standards are BSA, because colour formation depends on the protein (Smith et al. 1985, Anal Biochem 150:76–85). One kit's manual gives 20 to 2,000 µg/mL (standard protocol) and 5 to 250 µg/mL (enhanced protocol), the response nearly linear (Thermo Scientific Pierce BCA Protein Assay Kit, MAN0011430 Rev. D, read 10 October 2026). The zero-protein standard is the blank: assign it as a blank and subtract it.
Method
- Standard-curve interpolation. NIST/SEMATECH e-Handbook §2.3.6.6, inverse calibration; Cox et al., Assay Guidance Manual, Immunoassay Methods (2019), calibration and recovery. Linear least squares or the existing 4PL; mean responses per concentration; bounded interpolation. From A5 the 5PL and inverse-prediction intervals are their own methods (five-pl-calibration, calibration-inverse-prediction).Checked against exact rational least squares (linear, 7 cases) and SciPy 1.13.1 (4PL, 2 cases); out-of-range unknowns flagged (10 October 2026). R not run on the 4PL cases.
Limits
- For research and education only, and not medical advice: a C50 estimated here describes this assay under these conditions and is not a binding constant.