BCA Assay Calculator
Calculate protein concentration from BCA assay (bicinchoninic acid) absorbance data. Supports replicate averaging, blank subtraction, and unknown sample interpolation for Pre-loaded with standard BSA series (0–2000 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 BCA Protein Assay
The BCA (bicinchoninic acid) assay is a colorimetric method for determining total protein concentration. It is based on the reduction of Cu²⁺ to Cu⁺ by peptide bonds (biuret reaction), followed by chelation of Cu⁺ with BCA to produce a purple complex that absorbs at 562 nm. The standard working range is 20–2,000 μg/mL using BSA as the reference standard.
Replicate Measurements and Blank Subtraction
Running duplicate or triplicate measurements improves reliability. This calculator computes mean, SD, and CV% for each point. A CV% above 20% is flagged as high variability. 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 when your standards are within the assay's linear range (most common for BCA and Bradford). If R² drops below 0.99 with linear fitting, or if you are using an extended concentration range, try quadratic fitting. 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 absorbance falls outside the range covered by your standard points. The calculated concentration is based on extending the curve beyond measured data, which is unreliable. Dilute (or concentrate) your sample so that its absorbance 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.