Protein Standard Curve Calculator
Compute concentration from absorbance using a BCA or Bradford standard curve.
Concentrations and absorbance values must be listed in the same order, point by point.
Sample concentration
—µg/mL
Slope
4.9571e-4
Intercept
0.0024
Sensitivity
2,017.3
| Conc. (ug/mL) | Absorbance | Fitted | Residual |
|---|---|---|---|
| 0 | 0.000 | 0.002 | -0.002 |
| 250 | 0.125 | 0.126 | -0.001 |
| 500 | 0.252 | 0.250 | 0.002 |
| 750 | 0.374 | 0.374 | -0.000 |
| 1,000 | 0.505 | 0.498 | 0.007 |
| 1,500 | 0.741 | 0.746 | -0.005 |
Learn more
What it does
The protein standard curve calculator turns absorbance readings from a colorimetric protein assay into a concentration for your sample. You supply a set of standards (known concentrations) with their measured absorbance, and the tool fits a straight line that links concentration to signal. It then reads your unknown sample off that line. The same math works for BCA and Bradford assays, the difference is only in the chemistry and the useful range.
How it works
The standard curve is a linear regression of absorbance on concentration: A = slope x conc + intercept. To recover a concentration you invert it: conc = (A - intercept) / slope, where A is the sample absorbance. If the result comes out below zero it means the sample sits under the curve's intercept (less than the blank), so no concentration is reported. The undiluted concentration is the measured concentration multiplied by the dilution factor. Sensitivity is simply 1 / slope: how many ug/mL of protein correspond to one absorbance unit.
Worked example
With standards of 0, 250, 500, 750, 1000 and 1500 ug/mL giving absorbance of 0.000, 0.125, 0.252, 0.374, 0.505 and 0.741, the fit returns a slope of 4.9571e-4, an intercept of 0.00236 and R squared of 0.99977, with a sensitivity near 2017 ug/mL per absorbance unit. A sample reading 0.149 after a 10-fold dilution back-calculates to 295.8 ug/mL, which scales to 2958 ug/mL undiluted, or 2.96 mg/mL.
When to use it
Use this tool whenever you measure protein by a plate-based colorimetric assay and need the concentration of a lysate, purified fraction, or conditioned medium. It is most helpful when you must compare several samples on one curve, or when a sample lands near the edge of the standard range and you need to know whether the number is trustworthy. For BCA the usable interval is roughly 20-2000 ug/mL and for Bradford about 100-1400 ug/mL; outside those ranges dilute and re-measure rather than extrapolate.
FAQ
- Should I use BCA or Bradford for my protein assay?
- Choose BCA when your sample is in a detergent or reducing buffer that interferes with Bradford, or when you need the wider 20-2000 ug/mL range. Choose Bradford for a faster read and a sharper 100-1400 ug/mL working window. Both are read on the same plate reader, so the choice is mostly about buffer compatibility and your expected concentration.
- Why do I subtract the blank or use the zero-concentration point?
- The zero-concentration (blank) reading is the assay background from the reagent and the plate, not from your protein. Using it as the curve's intercept removes that offset so concentrations are measured relative to true zero. If you skip it, every sample reads slightly high by the size of the blank.
- How high does R squared need to be to trust the curve?
- Aim for R squared of at least 0.99 for a quantitative result. Values between 0.95 and 0.99 suggest the curve is usable for rough estimates but worth repeating, and below 0.95 the fit is too noisy for reliable concentrations. The tool flags any result under 0.99 so you can check pipetting or standards.
- What if my sample absorbance is outside the standard range?
- If the sample reads above the highest standard or below the lowest, the calculator extrapolates and shows a warning rather than a confident value. The right move is to dilute the sample into the calibrated range and re-measure, then apply the dilution factor. Extrapolated points should not be used for reporting.