How to Measure Protein Concentration at A280

Why proteins absorb at 280 nm

Tryptophan and tyrosine absorb ultraviolet light at 280 nm, and disulfide bonds add a little. Because the amount of each differs between proteins, every protein has its own extinction coefficient, and A280 readings are only as good as the coefficient you use.

The formula

From the Beer-Lambert law, concentration (M) = A280 ÷ (ε × path length), where ε is the molar extinction coefficient in M⁻¹ cm⁻¹. Multiply by the molecular weight for mg/mL. A shortcut is the Abs 0.1%, the absorbance of a 1 mg/mL solution, which is ε ÷ molecular weight: mg/mL = A280 ÷ Abs 0.1%.

Getting the extinction coefficient from the sequence

The method of Pace and colleagues (1995) counts the absorbing residues:

ε = 5,500 × Trp + 1,490 × Tyr + 125 × cystines

A cystine is a pair of cysteines joined by a disulfide bond; leave that term out for a reduced protein.

A worked example

Bovine serum albumin has ε = 43,824 M⁻¹ cm⁻¹ and a molecular weight of 66,430 Da, so its Abs 0.1% is 43,824 ÷ 66,430 = 0.66. A reading of 0.33 in a 1 cm cuvette is 0.33 ÷ 0.66 = 0.5 mg/mL, or 7.5 µM.

Pitfalls

• Blank with the same buffer the protein is in; imidazole, detergents and some reducing agents absorb at 280 nm.
• Nucleic acid contamination raises A280. A pure protein has an A260/A280 ratio around 0.6; values near 1 or above suggest DNA or RNA.
• Aggregates scatter light and read high. Spin or filter cloudy samples.
• Keep readings between about 0.1 and 1.0, diluting if needed.
• Proteins with no tryptophan or tyrosine cannot be measured this way; use a Bradford or BCA assay.

Calculators

The Protein Concentration Calculator works out the extinction coefficient from a pasted sequence, or takes a known coefficient or Abs 0.1%, and gives mg/mL and µM. The Beer-Lambert Law Calculator solves the general equation, and Protein Molecular Weight gives the mass from a sequence.