Home
Studies
& Data Analysis
Methods
Microscope studies
Flagella experiment
Laboratory math
Blood fractionation
Gel electrophoresis
Protein gel analysis
Mitochondria
Concepts/ theory
Keeping a lab notebook
Writing research papers
Dimensions & units
Using figures (graphs)
Examples of graphs
Experimental error
Representing error
Applying statistics
Principles of microscopy
Solutions & dilutions
Protein assays
Spectrophotometry
Fractionation & centrifugation
Radioisotopes and detection
Absorbance assays
Colorimetric assays
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
Visitors: to ensure that your message is not mistaken for SPAM, please include the acronym "Bios211" in the subject line of e-mail communications
Created by David R. Caprette (caprette@rice.edu), Rice University 24 May 95
Updated 10 Aug 12
Visitors: to ensure that your message is not mistaken for SPAM, please include the acronym "Bios211" in the subject line of e-mail communications
Created by David R. Caprette (caprette@rice.edu), Rice University 24 May 95
Updated 10 Aug 12