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List of methods
Direct absorbance measurementabsorbance at 280 nm Colorimetric assaysset up an assay |
Quantifying protein using absorbance at 280 nmConsiderations for useQuantifying protein by directly measuring absorbance is fast and convenient, since no additional reagents or incubations are required. No protein standard need be prepared and the procedure 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 measurements. Cell and tissue fractionation samples often contain insoluble or colored components that interfere. The most common use for this method is to monitor fractions from chromatography columns, or any time a quick estimation is needed and error in protein concentration is not a concern. This method is recommended for calibrating bovine serum albumin or other pure protein solutions for use as standards in other methods.PrincipleProteins 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.
EquipmentIn addition to standard liquid handling supplies
a spectrophotometer with UV lamp and quartz cuvette are required.
ProcedureCarry out steps 1-4 (280 nm only) for a very
rough estimate. Carry out all steps if nucleic acid contamination
is likely.
AnalysisUnknown 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) CommentsCold 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:
References
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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 |