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List of methods

Direct absorbance measurement

absorbance at 280 nm
absorbance at 205 nm
extinction coefficient

Colorimetric assays

set up an assay
spectrophotometry
modified Lowry

biuret
Bradford
Bicinchoninic Acid (Smith)

Determining an Extinction Coefficient for a Protein of Unknown Concentration

Considerations for use

The concentration can be determined for a solution of a pure protein with unknown extinction coefficient.

Principle

See the discussion for quantifying protein by measuring absorbance at 280 nm.

Equipment

In addition to standard liquid handling supplies a spectrophotometer with UV lamp and quartz cuvette are required.

Procedure

Dilute the solution about 30 fold for the reading at 205 nm and include 0.01% Brij 35 in the buffer to prevent adsorption of protein onto plastic or glass surfaces.

Analysis

Use the following formula to determine the extinction coefficient at 205 nm:

E(205 nm) = 27 + 120 x (A280 divided by A205)

The reading at 205 nm must be multiplied by the dilution factor before using the formula. Next, determine protein concentration:

Protein concentration (M) = A205 divided by E(205 nm)

You can now determine the extinction coefficient for 280 nm:

E(280 nm) = concentration (M) divided by A280

Comments

An abnormal phenylalanine content will throw off the result considerably. The accuracy of the technique depends on an average amino acid composition.

References

  • Scopes, RK. Analytical Biochemistry 59: 277. 1974.
  • Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69. 1990.

Copyright and Intended Use
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Created by David R. Caprette (caprette@rice.edu), Rice University 24 May 95
Updated 12 Jun 15