Absorbance Assay (280 nm)
Considerations for use
Absorbance assays are fast and convenient, since no additional
reagents or incubations are required. No protein standard need be prepared.
The assay does not consume the protein. The relationship of absorbance to
protein concentration is linear. Because different proteins and nucleic acids
have widely varying absorption characteristics there may be considerable
error, especially for unknowns or protein mixtures. Any non-protein component
of the solution that absorbs ultraviolet light will intefere with the assay.
Cell and tissue fractionation samples often contain insoluble or colored components
that interfere. The most common use for the absorbance assay is to monitor
fractions from chromatography columns, or any time a quick estimation is needed
and error in protein concentration is not a concern. An absorbance assay
is recommended for calibrating bovine serum albumin or other pure protein
solutions for use as standards in other methods.
Principle
Proteins in solution absorb ultraviolet light with absorbance
maxima at 280 and 200 nm. Amino acids with aromatic rings are the primary
reason for the absorbance peak at 280 nm. Peptide bonds are primarily responsible
for the peak at 200 nm. Secondary, tertiary, and quaternary structure all
affect absorbance, therefore factors such as pH, ionic strength, etc. can
alter the absorbance spectrum.
Equipment
In addition to standard liquid handling supplies a spectrophotometer
with UV lamp and quartz cuvette are required.
Procedure
Carry out steps 1-4 (280 nm only) for a very rough estimate.
Carry out all steps if nucleic acid contamination is likely.
- Warm up the UV lamp (about 15 min.)
- Adjust wavelength to 280 nm
- Calibrate to zero absorbance with buffer solution only
- Measure absorbance of the protein solution
- Adjust wavelength to 260 nm
- Calibrate to zero absorbance with buffer solution only
- Measure absorbance of the protein solution
Analysis
Unknown proteins or protein mixtures. Use the following
formula to roughly estimate protein concentration. Path length for most spectrometers
is 1 cm.
Concentration (mg/ml) = Absorbance at 280 nm divided by path
length (cm.)
Pure protein of known absorbance coefficient. Use
the following formula for a path length of 1 cm. Concentration is in mg/ml,
%, or molarity depending on which type coefficient is used.
concentration = Absorbance at 280 nm divided by absorbance
coefficient
To convert units, use these relationships:
Mg protein/ml = % protein divided by 10 = molarity divided
by protein molecular weight
Unknowns with possible nucleic acid contamination.
Use the following formula to estimate protein concentration:
Concentration (mg/ml) = (1.55 x A280) - 0.76 x A260)
Comments
Cold solutions can fog up the cuvette, while warm solutions
can release bubbles and interfere with the readings. For concentrated solutions
(absorbance greater than 2) simply dilute the solution.
Absorbance coefficients of some common protein standards:
- Bovine serum albumin (BSA): 63
- Bovine, human, or rabbit IgG: 138
- Chicken ovalbumin: 70
References
- Layne, E. Spectrophotometric and Turbidimetric Methods for Measuring
Proteins. Methods in Enzymology 3: 447-455. 1957.
- Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182:
50-69. 1990.
