Prepare BSA standards at known concentrations (typically 25-2000 µg/mL for BCA), measure absorbance at 562 nm in duplicate or triplicate, then enter the data into this calculator. The tool fits a model (linear, quadratic, or 4PL) and back-calculates unknown concentrations. A good curve should have R² above 0.99.

Linear works for narrow ranges (e.g. 125-1000 µg/mL in BCA). Quadratic handles moderate curvature. 4PL captures the full sigmoidal response including saturation, making it the most robust for the full BCA range. Bradford is more non-linear, so quadratic or 4PL is recommended. Use the "Auto" setting to let the calculator pick the best model.

R² should be above 0.99 for BCA and Bradford assays, ideally above 0.995. Below 0.98 suggests pipetting errors, reagent issues, or incubation problems. Each standard should back-calculate within 80-120% of its nominal value, and replicate %CV should be below 10% for BCA or 15% for Bradford.

Outliers are flagged when replicate %CV exceeds your threshold (default 20%). Common causes include air bubbles, pipetting errors, or plate edge effects. This calculator identifies outlier replicates using the >2 SD criterion and shows the impact on the curve fit. Best practice: exclude the outlier replicate, not the entire standard point, then re-fit.

BCA measures peptide bond reduction of Cu²⁺ at 562 nm (20-2000 µg/mL range, 30 min incubation, detergent-compatible). Bradford measures Coomassie dye binding at 595 nm (1-1400 µg/mL, 5 min, sensitive to detergents). Lowry reads at 750 nm (200-1500 µg/mL). Choose BCA for general quantification, Bradford for speed, and Micro BCA for dilute samples (0.5-20 µg/mL).

Multiply the back-calculated concentration by the dilution factor. For example, a 1:10 dilution with a back-calculated 150 µg/mL gives an actual concentration of 1500 µg/mL. Add dilution factors in the unknowns input using the 'x' prefix (e.g. x10). Always dilute so absorbance falls within the standard range.