Protein Concentration Calculator

Determine protein concentration from absorbance at 280nm using the Beer-Lambert law. Enter your spectrophotometer readings for instant calculation.

Measured absorbance value

Protein-specific extinction coefficient

Protein molecular weight

Usually 1 cm for standard cuvettes

c = A / (ε × l), where c = concentration (mol/L), A = absorbance at 280nm, ε = extinction coefficient (L·mol⁻¹·cm⁻¹), l = pathlength (cm); Concentration (mg/mL) = c × Molecular Weight × 1000
For A280 = 0.650, ε = 43,824 L·mol⁻¹·cm⁻¹, MW = 66,430 g/mol, l = 1 cm: Molar concentration = 1.483 × 10⁻⁵ mol/L; Concentration = 0.985 mg/mL; Concentration = 14.83 μM

How is protein concentration measured using absorbance?

Protein concentration is measured using the Beer-Lambert Law: A = εcl, where A is absorbance at 280nm, ε is the extinction coefficient (L·mol⁻¹·cm⁻¹), c is concentration (mol/L), and l is pathlength (cm). Most spectrophotometers use a 1 cm pathlength. Proteins absorb UV light at 280nm due to aromatic amino acids (tryptophan, tyrosine, and phenylalanine). By measuring absorbance and knowing the extinction coefficient, you can calculate concentration.

What is the extinction coefficient and where do I find it?

The extinction coefficient (ε) is a measure of how strongly a protein absorbs light at 280nm. It depends on the protein's amino acid sequence, specifically the number of tryptophan, tyrosine, and cysteine residues. You can find it in the protein's literature, calculate it using tools like ProtParam from ExPASy, or estimate it: typical values range from 10,000 to 200,000 L·mol⁻¹·cm⁻¹. For BSA (bovine serum albumin), ε ≈ 43,824 L·mol⁻¹·cm⁻¹.

What is a good absorbance reading for protein samples?

Ideal absorbance readings at A280 are between 0.1 and 1.0 for accurate measurements. Below 0.1: too dilute, consider concentrating your sample. Above 1.0: too concentrated, dilute your sample and recalculate. The 260/280 ratio indicates purity: pure protein ≈ 0.6, protein with nucleic acid contamination > 0.6 (nucleic acids absorb at 260nm). A ratio of 1.8 at A260/A280 indicates pure DNA, while 2.0 indicates pure RNA.

How do I dilute my protein sample to the correct concentration?

Use the dilution formula: C1V1 = C2V2, where C1 is initial concentration, V1 is volume to take, C2 is desired concentration, and V2 is final volume. For example, to dilute a 10 mg/mL stock to 1 mg/mL in 1 mL total: V1 = (C2 × V2) / C1 = (1 × 1000) / 10 = 100 μL. Add 100 μL of stock to 900 μL of buffer. Always mix thoroughly after dilution and verify concentration with a new absorbance reading.