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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 the 280 nm absorbance assay.

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 10 Aug 12