Bradford Assay Calculator
Calculate protein concentration from Bradford assay (Coomassie Blue dye binding) absorbance data. Supports replicate averaging, blank subtraction, and unknown sample interpolation for Pre-loaded with standard BSA series (0–25 μg/mL).
Standard Data
📋 Paste from spreadsheet
| ✓ | Conc. | Rep 1 | Rep 2 | Rep 3 |
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Unknown Samples
📋 Paste from spreadsheet
| Name | Rep 1 | Rep 2 | Rep 3 | Dilution Factor |
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About the Bradford Protein Assay
The Bradford assay uses Coomassie Brilliant Blue G-250 dye, which binds to protein under acidic conditions. The dye shifts from reddish-brown to blue upon binding, measured at 595 nm. Typical working ranges depend on assay format and reference protein — commonly ~1–25 μg/mL for micro-format and ~100–1,500 μg/mL for standard-format protocols.
Replicate Measurements and Blank Subtraction
Running duplicate or triplicate measurements improves reliability. This calculator computes mean, SD, and CV% for each point (CV thresholds are practical heuristics, not universal acceptance criteria). In Auto blank mode, the 0 μg/mL standard absorbance is subtracted from all readings.
ℹ️ Frequently Asked Questions
When should I use linear vs quadratic fitting?
Use linear fitting for data within the assay's validated linear range (most common for BCA and Bradford). If you observe reproducible curvature across an extended calibrated range, consider quadratic fitting. Before switching models, first check for outliers, pipetting errors, and whether the standards exceed the assay's working range. A good quadratic fit will have R² > 0.995 and a small quadratic coefficient (a).
What does "extrapolation" mean and why is it warned?
Extrapolation occurs when an unknown sample's result falls outside the calibrated range — either the absorbance is outside the standard signal range, or the calculated concentration is outside the standard concentration range. Extrapolated results are unreliable. Dilute (or concentrate) your sample so that its reading falls within the standard range.
How do I handle outlier standard points?
Click the checkbox next to any standard point to exclude it from the fitting. The excluded point will appear grayed out on the chart with an × marker. The curve and R² will be recalculated using only the included points. Document which points you excluded and why in your lab notebook.
What R² value is acceptable?
For most colorimetric protein assays, R² ≥ 0.99 is considered acceptable. Below 0.98, the curve may not be reliable for accurate quantification. Check for pipetting errors, expired reagents, or standards that are outside the assay's working range.
What is the dilution factor?
If you diluted your sample before measurement (e.g., 1:5 dilution), enter 5 as the dilution factor. The calculator will multiply the curve-derived concentration by this factor to give the original sample concentration. A dilution factor of 1 means no dilution was performed.