Determining an extinction coefficient for a protein



Home	Laboratory Studies	Recordkeeping, Writing, & Data Analysis	Laboratory Methods
	Overview Microscope studies Flagella experiment Laboratory math Blood fractionation Gel electrophoresis Protein gel analysis Mitochondria Concepts/ theory	Overview Keeping a lab notebook Writing research papers Dimensions & units Using figures (graphs) Examples of graphs Experimental error Representing error Applying statistics	Overview Principles of microscopy Solutions & dilutions Protein assays Spectrophotometry Fractionation & centrifugation Radioisotopes and detection

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 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 12 Jun 15