Protein Concentration Calculator

When measuring protein samples with a spectrophotometer, the Protein Concentration Calculator converts your absorbance at 280 nm into an actual concentration using the Beer-Lambert law. Enter your A280 reading, molar extinction coefficient, molecular weight, cuvette pathlength, and dilution factor to get your protein concentration in mg/mL. Secondary outputs include molar concentration (µM), concentration in µg/µL, and corrected absorbance after blank subtraction.

Measured absorbance at 280 nm wavelength

Background absorbance to subtract

M⁻¹·cm⁻¹

Protein-specific extinction coefficient at 280 nm

Da

Molecular weight of the protein in Daltons

cm

Length of light path through sample (standard cuvette = 1 cm)

Factor by which sample was diluted (1 = no dilution)

Results

Protein Concentration

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Molar Concentration

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Concentration (µg/µL)

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Corrected Absorbance

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Frequently Asked Questions

How do I calculate protein concentration from A280 absorbance?

Use the Beer-Lambert law: C = A/(ε × l), where C is concentration, A is absorbance at 280nm, ε is the molar extinction coefficient, and l is pathlength. Convert to mg/mL by multiplying by molecular weight and appropriate unit conversions.

What is the typical extinction coefficient for proteins at 280nm?

Extinction coefficients vary widely by protein, typically ranging from 5,000 to 200,000 M⁻¹·cm⁻¹. BSA has an extinction coefficient of ~43,824 M⁻¹·cm⁻¹. Check protein databases or literature for your specific protein.

Why do I need to subtract blank absorbance?

Blank correction removes background absorbance from the buffer, cuvette, and instrument baseline. This ensures your measurement reflects only the protein contribution to the total absorbance.

What pathlength should I use for standard cuvettes?

Standard spectrophotometer cuvettes have a 1 cm pathlength. Micro-cuvettes or specialized cells may have different pathlengths (0.1-10 cm). Always check your cuvette specifications.

How does dilution factor affect the final concentration?

If you diluted your sample before measurement, multiply the calculated concentration by the dilution factor to get the original concentration. For example, a 1:10 dilution has a dilution factor of 10.

What are the limitations of A280 protein quantification?

A280 measurements can be affected by nucleic acid contamination, turbidity from aggregates, and proteins with few aromatic residues. The method works best for pure protein solutions.

How accurate is protein concentration measurement at 280nm?

A280 measurements are typically accurate within 5-10% for pure proteins when using the correct extinction coefficient. Accuracy decreases with contamination or if using estimated extinction coefficients.

Can I use this calculator for protein mixtures?

This calculator is designed for single proteins with known extinction coefficients. For protein mixtures, consider using colorimetric assays like Bradford or BCA that don't require specific extinction coefficients.