Enter your Absorbance at 562 nm, Standard Curve Slope/Intercept, and Dilution Factor into the BCA Assay Calculator to find your Protein Concentration in µg/mL and g/L, with optional Molar Concentration when you provide the Molecular Weight.
Protein Concentration
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Concentration (µg/mL)
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Concentration (g/L)
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Molar Concentration
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The BCA (bicinchoninic acid) assay is a colorimetric protein quantification method that measures absorbance at 562 nm. Proteins reduce Cu²⁺ to Cu¹⁺ under alkaline conditions, and the Cu¹⁺ ions form a purple complex with BCA reagent, creating a linear response proportional to protein concentration.
Prepare BSA standards ranging from 0-2000 µg/mL, run them through the BCA protocol, and plot absorbance vs concentration. The resulting linear equation (y = mx + b) gives you the slope and intercept values needed for this calculator.
If absorbance exceeds 2.0 AU or falls outside your standard curve range, dilute your sample and increase the dilution factor accordingly. The BCA assay works best within 0.1-2.0 AU range for accurate quantification.
The blank (buffer without protein) accounts for background absorbance from reagents and buffer components. Subtracting the blank from sample readings ensures accurate protein concentration calculations by removing non-specific absorbance.
The dilution factor represents how much you diluted your original sample. For example, if you diluted 1:10, enter 10 as the dilution factor. The calculator multiplies the calculated concentration by this factor to report the original sample concentration.
Common interferences include reducing agents (DTT, TCEP), high concentrations of sugars, lipids, or nucleic acids. Unlike Bradford assay, BCA is compatible with most detergents and denaturants but may show interference with copper-chelating agents.
BCA assay typically shows ±5-10% accuracy with good linearity across a wide range (20-2000 µg/mL). It's more tolerant of interfering substances than Bradford assay but less rapid, requiring 30-60 minute incubation times.
Molecular weight is needed to convert mass concentration (mg/mL) to molar concentration (µM). This is useful for enzymatic studies, binding assays, or when calculating protein stoichiometry in biochemical reactions.