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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PROTEIN QUANTITATION

DETERMINATION OF PROTEIN CONCENTRATION BY UV ABSORPTION

Determination of Protein Concentration by UV Absorption
Contributor: The Laboratory of Michael Chamberlin at the University of California, Berkeley
 
Overview
Most proteins exhibit a distinct ultraviolet light absorption maximum at 280 nm wavelength, due primarily to the presence of Tyrosine and Tryptophan. Since the Tyrosine and Tryptophan content of various enzymes varies within narrow limits, the absorption peak at 280 nm has been used as a rapid and fairly sensitive measure of protein concentration. Unfortunately, nucleic acids, which are apt to be present in enzyme preparations, absorb light at wavelength 280 nm. Nucleic acids, however, absorb much more strongly at 260 nm wavelength UV light. For protein it is the reverse situation. This assay uses this reverse relationship to calculate the interference of nucleic acids in the estimation of protein concentration.
 
Procedure
1. Place 1 ml of diluted protein solution in a quartz cuvette and measure the absorbance at both 280 nm and 260 nm in a spectrophotometer.

2. Calculate the 280 nm:260 nm ratio and then determine the corresponding factor (F) from the table given below.

3. Substitute the measured values in the following equation:

Protein Concentration (mg/ml) = F X Absorbance at 280 nm X 1/d

Where d is the cuvette width in cm (see Hint #1).

R280/R260, Nucleic Acid %, F

1.75, 0.00, 1.116

1.63, 0.25, 1.081

1.52, 0.50, 1.054

1.40, 0.75, 1.023

1.36, 1.00, 0.994

1.30, 1.25, 0.970

1.25, 1.50, 0.944

1.16, 2.00, 0.899

1.09, 2.50, 0.852

1.03, 3.00, 0.814

0.979, 3.50, 0.776

0.939, 4.00, 0.743

0.874, 5.00, 0.682

0.846, 5.50, 0.656

0.822, 6.00, 0.632

0.804, 6.50, 0.607

0.784, 7.00, 0.585

0.767, 7.50, 0.565

0.753, 8.00, 0.545

0.730, 9.00, 0.508

0.705, 10.00, 0.478

0.671, 12.00, 0.422

0.644, 14.00, 0.377

0.615, 17.00, 0.322

0.595, 20.00, 0.278

Solutions
This bioProtocol does not use any solutions
 
BioReagents and Chemicals
This bioProtocol does not use any reagents
 
Protocol Hints
1. The concentration of the protein in solution is calculated according to the treatment of Warburg and Christian, who have determined the extinction coefficient of crystalline yeast enolase and of yeast acid. With this protocol, it is not necessary to know the nucleic acid concentration of the sample to find the protein concentration of an unknown sample. This method gives considerable error with mixtures containing more than 20% nucleic acids or with very turbid solutions.

 
Citation and/or Web Resources
1. Layne E. Spectrophotometric and turbidometric methods for measuring proteins. Meth. Enzymol. 1957;Vol 3:447