How it works
Pick the assay and its usual standards are filled in, or enter your own. Paste the standards' readings and your samples' readings, open a file, or read the whole plate through the plate layout. The blank, the 0 standard or blank wells on the plate, is subtracted from every reading, the standards are fitted, and each sample is read off the curve from the mean of its replicates and multiplied by the sample dilution.
Which curve to fit
Bradford and BCA responses bend over at higher concentrations, so a straight line through all the standards reads samples high in the middle of the range and low at the ends. A quadratic follows the bend and is the default for most assays here. A linear fit is fine for the micro assays, or over the straight part of a curve. Samples are only read within the range of the standards; anything above it should be diluted and measured again.
Worked example
The example loaded on this page is a standard Bradford assay at 595 nm with BSA standards from 125 to 2000 µg/mL in duplicate, and three samples diluted 1:5. After subtracting the 0 standard, a quadratic fit gives R² = 0.9998, and Lysate 1, with a blank-subtracted mean reading of 0.4580, reads 705.0 µg/mL from the curve: 3525 µg/mL in the undiluted sample.
Choosing a standard
Proteins give different amounts of colour in these assays, so results are relative to the standard. BSA is the usual choice. Antibodies give noticeably less colour than BSA in a Bradford assay, so a bovine gamma globulin (BGG) standard gives closer values for them, and purified antibodies are better measured by A280. BCA varies less between proteins than Bradford, but is affected by reducing agents and chelators in the buffer.
For your records
Download the chart and samples table as one figure, in PNG for a lab notebook or report, or SVG to keep it sharp at any size. Figures are always on a white background, whatever the screen theme.
Accuracy
The fit and the back-calculated sample concentrations are checked against an independent least-squares calculation before every release.
This calculator is checked against independent calculations before every release. How the calculators are checked.
References
- Bradford MM (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248–254.
- Smith PK et al. (1985). Measurement of protein using bicinchoninic acid. Analytical Biochemistry 150: 76–85.
- Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193: 265–275.