Enter your Absorbance at 280 nm, Molar Extinction Coefficient, and Pathlength into the Protein Concentration Calculator to apply Beer-Lambert law and find your protein concentration — with optional Dilution Factor and Molecular Weight for molar concentration.
Protein Concentration
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Molar Concentration
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Concentration
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Concentration
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Use the Beer-Lambert law: C = A / (ε × l), where C is concentration, A is absorbance at 280 nm, ε is the molar extinction coefficient, and l is pathlength. Multiply by dilution factor if your sample was diluted.
The extinction coefficient (ε) is a protein-specific value that indicates how strongly it absorbs light at 280 nm. You can find it in protein databases like UniProt, calculate it from amino acid composition, or use typical values (e.g., BSA: 43,824 M⁻¹·cm⁻¹).
Create a standard curve using known protein concentrations, measure your sample signal (absorbance/fluorescence), then use the curve equation C = (signal - intercept) / slope to determine concentration.
Most standard spectrophotometer cuvettes have a 1 cm pathlength. Microplate readers typically use shorter pathlengths (0.2-0.6 cm). Check your instrument specifications or cuvette documentation.
Multiply your calculated concentration by the dilution factor. For example, if you diluted your sample 1:10 before measurement, use a dilution factor of 10 to get the original sample concentration.
A blank (buffer without protein) accounts for background absorbance from your buffer, salts, or other components. Subtracting the blank gives you the true protein absorbance signal.
A280 measurements can be affected by nucleic acid contamination, requires knowing the extinction coefficient, and works best for proteins with aromatic amino acids (Trp, Tyr, Phe). Consider alternative methods like Bradford or BCA assays for unknown proteins.