Absorbance Assay (280 nm)

Considerations for use

Absorbance assays are fast and convenient, since no additional reagents or incubations are required. No protein standard need be prepared. The assay does not consume the protein. The relationship of absorbance to protein concentration is linear. Because different proteins and nucleic acids have widely varying absorption characteristics there may be considerable error, especially for unknowns or protein mixtures. Any non-protein component of the solution that absorbs ultraviolet light will intefere with the assay. Cell and tissue fractionation samples often contain insoluble or colored components that interfere. The most common use for the absorbance assay is to monitor fractions from chromatography columns, or any time a quick estimation is needed and error in protein concentration is not a concern. An absorbance assay is recommended for calibrating bovine serum albumin or other pure protein solutions for use as standards in other methods.

Principle

Equipment

Procedure

Analysis

Concentration (mg/ml) = Absorbance at 280 nm divided by path length (cm.)

Pure protein of known absorbance coefficient. Use the following formula for a path length of 1 cm. Concentration is in mg/ml, %, or molarity depending on which type coefficient is used.

concentration = Absorbance at 280 nm divided by absorbance coefficient

To convert units, use these relationships:

Mg protein/ml = % protein divided by 10 = molarity divided by protein molecular weight

Unknowns with possible nucleic acid contamination. Use the following formula to estimate protein concentration:

Concentration (mg/ml) = (1.55 x A280) – 0.76 x A260)

Comments

Absorbance coefficients of some common protein standards:

References