& Data Analysis
Protein gel analysis
|List of methods
Direct absorbance measurement
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.
See the discussion for quantifying protein by measuring absorbance at 280 nm.
In addition to standard liquid handling supplies a spectrophotometer with UV lamp and quartz cuvette are required.
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.
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
An abnormal phenylalanine content will throw off the result considerably. The accuracy of the technique depends on an average amino acid composition.