measuring protein concentration using absorbance at 280 nm.pdf

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11/10/13 1:Measuring protein concentration using absorbance at 280 nm

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Absorbance assays

absorbance at 280nmabsorbance at 205nmextinctioncoefficient

Colorimetric assays

set up an assayspectrophotometrymodified LowrybiuretBradfordBicinchoninicAcid (Smith)

Absorbance Assay (280 nm)Considerations for use

Absorbance assays are fast and convenient, since no additional reagents orincubations are required. No protein standard need be prepared. The assay doesnot consume the protein. The relationship of absorbance to protein concentration linear. Because different proteins and nucleic acids have widely varyingabsorption characteristics there may be considerable error, especially forunknowns or protein mixtures. Any non-protein component of the solution that

absorbs ultraviolet light will intefere with the assay. Cell and tissue fractionationsamples often contain insoluble or colored components that interfere. The mostcommon use for the absorbance assay is to monitor fractions from chromatographcolumns, or any time a quick estimation is needed and error in proteinconcentration is not a concern.An absorbance assay is recommended forcalibrating bovine serum albumin or other pure protein solutions for use asstandards in other methods.
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Principle

Proteins in solution absorb ultraviolet light with absorbance maxima at 280 and200 nm. Amino acids with aromatic rings are the primary reason for theabsorbance peak at 280 nm. Peptide bonds are primarily responsible for the peakat 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 lamand 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.

1. Warm up the UV lamp (about 15 min.)2. Adjust wavelength to 280 nm3. Calibrate to zero absorbance with buffer solution only4. Measure absorbance of the protein solution5. Adjust wavelength to 260 nm6. Calibrate to zero absorbance with buffer solution only7. Measure absorbance of the protein solution

AnalysisUnknown proteins or protein mixtures.Use the following formula to roughlyestimate 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 apath length of 1 cm. Concentration is in mg/ml, %, or molarity depending onwhich 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 molecularweight

Unknowns with possible nucleic acid contamination.Use the following formula t


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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 bubblesand interfere with the readings. For concentrated solutions (absorbance greaterthan 2) simply dilute the solution.

Absorbance coefficients of some common protein standards:

Bovine serum albumin (BSA): 63Bovine, human, or rabbit IgG: 138Chicken ovalbumin: 70

References

Layne, E. Spectrophotometric and Turbidimetric Methods for MeasuringProteins.Methods in Enzymology 3:447-455. 1957.Stoscheck, CM. Quantitation of Protein.Methods in Enzymology 182:50-69. 1990.

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line of e-mail communicationsCreated by David R. Caprette ([email protected]), Rice University 24 May 95

Updated 10 Aug 12
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measuring protein concentration using absorbance at 280 nm.pdf

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