What it does
A protein assay turns protein into colour. In a Bradford assay Coomassie dye shifts to blue when it binds protein (read near 595 nm); in a BCA assay protein reduces copper, which then makes a purple colour with BCA (read near 562 nm). You measure a series of standards of known concentration, usually BSA, draw a standard curve and read your samples off it. This page does that in one go:
- Blank subtraction. The mean of the 0 standard (or a blank you type) is taken away from every reading.
- Three curves. Straight line is fine for a narrow range or a BCA assay. Quadratic follows the bend at the top of a Bradford curve. Point to point joins the standards’ means with straight segments and assumes no shape at all.
- Replicates. Each standard and sample can have any number of readings. You get the mean, the SD and the CV, and samples whose CV is above your limit are flagged.
- Dilutions. Each sample has its own dilution factor, so the page reports both the concentration in the assay and in the original sample.
- Range checks. Samples above the top standard, below the lowest standard, past the top of a quadratic, or at the blank are flagged in the table.
- Loading volumes. Type an amount (20 µg for an SDS-PAGE lane, say) and an optional final volume, and the page gives the µL of each sample and of buffer to add.
How to use it
- Standards. One line per standard: the concentration, then each absorbance. Paste straight from a plate reader export or spreadsheet. A header line is fine.
- Settings. Choose the concentration unit, the curve and how the blank is handled.
- Samples. One line per sample: a name, the dilution factor (10 or 1:10 for one part sample in ten), then each absorbance.
- Read the results. Check the curve picture and the standards read back table, then the samples table. Download the CSV or a PNG, PDF or Word report.
Worked example
The example is a made-up Bradford assay: eight BSA standards from 0 to 2000 µg/mL in triplicate, and four samples. The blank (the 0 standard) is 0.4517.
With the quadratic curve, R² is 0.99956 and every standard reads back within 2 % of its value. The cell lysate, diluted 10-fold, has a mean absorbance of 0.8677, a net absorbance of 0.4160, reads 590 µg/mL in the assay and so 5900 µg/mL in the lysate (SD 104 from its three readings). To load 20 µg you need 3.39 µL, topped up with 21.6 µL of buffer to 25 µL.
The other samples show the flags: the tissue extract has one stray reading (CV 11.7 %, above the 10 % limit), the purified protein reads 2751 µg/mL, above the top standard (dilute it and measure again), and the wash fraction reads 31.99 µg/mL, below the lowest standard of 100.
Switch to the straight line and R² drops to 0.98264; the 100 µg/mL standard reads back as only 35.04 µg/mL (35.0 %) and the lysate comes out at 6666 µg/mL, 13 % too high. That is why a straight line through a whole Bradford range is a poor idea. Point to point gives 5868 µg/mL for the lysate.
Tips
- Make the standards in the same buffer as your samples; detergents and reducing agents change the colour.
- Dilute samples so they land in the middle of the curve, where it is steepest and most precise.
- Run at least duplicates, better triplicates, of standards and samples.
- Treat the result as relative to BSA. Different proteins give different amounts of colour per µg.
FAQ
Which curve should I use? For a Bradford assay over a wide range, the quadratic or point to point. For BCA or a narrow range, the straight line is often enough. Look at the standards read back table: a good curve reads every standard back within about 10 %.
Why is the curve fitted to every reading and not the means? So that standards with more replicates count more and the scatter is real. With the same number of replicates at each level the line is identical.
Why is the zero standard not flagged as “below the lowest standard”? Ranges are judged against the lowest standard above zero, because at the bottom of a curve readings are least reliable.
Are my readings sent anywhere? No. Everything is worked out on this page and nothing is stored.
Where the numbers come from
- Blank subtraction, the least-squares straight line and quadratic, point-to-point reading, mean, SD and CV are standard methods, written by fibub and checked in the tests against NumPy (polyfit, roots, interp, std) and against curves worked by hand.
- The example standards and sample readings are made up for this page. They are not taken from any kit insert, paper or database.
Standard scientific values, such as constants, atomic weights and melting points, are facts found in every textbook; we name where ours were checked. If you are an author or publisher and want a value, a credit or a table changed or removed, tell us and we will act quickly.
Last updated 2026-11-09.