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)

Quantifying protein using absorbance at 205 nm

Considerations for use

See considerations listed under the absorbance at 280 nm. This method is just as convenient. It may be preferred if there is excessive contamination by nucleic acids, since nucleic acids absorb very little radiation at 205 nm. Setting the wavelength is a bit tricky since 205 nm is right on the shoulder of the protein peak.

Principle

See the discussion for quantifying protein using absorbance at 280 nm.

Equipment

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

Procedure

Include 0.01% Brij 35 in the buffer to prevent adsorption of protein onto plastic or glass surfaces. This is necessary for measurements at 205 nm because losses are proportionately higher in dilute solutions.

Warm up the UV lamp (about 15 min.)
Adjust wavelength to 205 nm
Calibrate to zero absorbance with buffer solution only
Measure absorbance of the protein solution

Analysis

Protein concentration (mg/ml) = (Absorbance at 205 nm)/31.

Comments

Cold solutions can fog up the cuvette, while warm solutions can release bubbles and interfere with the readings. Solutions must be much more dilute than for measurements at 280 nm. Proteins absorb much more strongly at 205 nm, and there is supposedly less variability from protein to protein. In addition to the need for an accurate wavelength setting, stray light can be a major problem. To avoid these problems, use a 10 microgram/microliter solution of bovine serum albumin as a standard. With buffer blank as zero absorbance, determine the concentration of an unknown (concentration between 0 and 10 micrograms/microliter) by interpolation. This is acceptable because of the linear relationship of absorbance and concentration in the 0 to 10 microgram/microliter range.

The problem of an accurate wavelength setting can be avoided by determining absorbance at 210 nm (extinction coefficients range from 20 to 24). However there is less sensitivity and more variation with buffer conditions.

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



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)	Quantifying protein using absorbance at 205 nm Considerations for use See considerations listed under the absorbance at 280 nm. This method is just as convenient. It may be preferred if there is excessive contamination by nucleic acids, since nucleic acids absorb very little radiation at 205 nm. Setting the wavelength is a bit tricky since 205 nm is right on the shoulder of the protein peak. Principle See the discussion for quantifying protein using absorbance at 280 nm. Equipment In addition to standard liquid handling supplies a spectrophotometer with UV lamp and quartz cuvette are required. Procedure Include 0.01% Brij 35 in the buffer to prevent adsorption of protein onto plastic or glass surfaces. This is necessary for measurements at 205 nm because losses are proportionately higher in dilute solutions. Warm up the UV lamp (about 15 min.) Adjust wavelength to 205 nm Calibrate to zero absorbance with buffer solution only Measure absorbance of the protein solution Analysis Protein concentration (mg/ml) = (Absorbance at 205 nm)/31. Comments Cold solutions can fog up the cuvette, while warm solutions can release bubbles and interfere with the readings. Solutions must be much more dilute than for measurements at 280 nm. Proteins absorb much more strongly at 205 nm, and there is supposedly less variability from protein to protein. In addition to the need for an accurate wavelength setting, stray light can be a major problem. To avoid these problems, use a 10 microgram/microliter solution of bovine serum albumin as a standard. With buffer blank as zero absorbance, determine the concentration of an unknown (concentration between 0 and 10 micrograms/microliter) by interpolation. This is acceptable because of the linear relationship of absorbance and concentration in the 0 to 10 microgram/microliter range. The problem of an accurate wavelength setting can be avoided by determining absorbance at 210 nm (extinction coefficients range from 20 to 24). However there is less sensitivity and more variation with buffer conditions. 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