Protein Determination by UV Absorption
Quantitation of the amount of protein in a solution is possible in a simple spectrometer. Absorption of radiation in the near UV by proteins depends on the Tyr and Trp content (and to a very small extent on the amount of Phe and disulfide bonds). Therefore the A280 varies greatly between different proteins (for a 1 mg/mL solution, from 0 up to 4 [for some tyrosine-rich wool proteins], although most values are in the range 0.5-1.5 ). The advantages of this method are that it is simple, and the sample is recoverable. The method has some disadvantages, including interference from other chromophores, and the specific absorption value for a given protein must be determined. The extinction of nucleic acid in the 280-nm region may be as much as 10 times that of protein at their same wavelength, and hence, a few percent of nucleic acid can greatly influence the absorption.
KeywordsExtinction Coefficient Potassium Phosphate Buffer Protein Solution Sodium Chloride Solution Quartz Cuvets
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
Kirschenbaum, D. M. (1975) Molar absorptivity and A1%/1 cm values for proteins at selected wavelengths of the ultraviolet and visible regions. Analyt. Biochem.
, 465–84.PubMedCrossRefGoogle Scholar
Scopes, R. K. (1974) Measurement of protein by spectrometry at 205 nm. Analyt. Biochem.
, 277–282.PubMedCrossRefGoogle Scholar
Goldfarb, A. R., Saidel, L. J., and Mosovich, E. (1951) The ultraviolet absorption spectra of proteins. J. Biol. Chem.
193, 397–04.PubMedGoogle Scholar
Gill, S. C. and von Hippel, P. H. (1989) Calculation of protein extinction coefficients from amino acid sequence data. Analyt. Biochem.
, 319–326.PubMedCrossRefGoogle Scholar
Waddell, W. J. (1956) A simple UV spectrophotometric method for the determination of protein. J. Lab. Clin. Med.
, 311–314.PubMedGoogle Scholar
Layne, E. (1957) Spectrophotornetric and turbidimetric methods for measuring proteins. Meth. Enzymol.
, 447–54.CrossRefGoogle Scholar
Whitaker, J. R. and Granum, P. E. (1980) An absolute method for protein determination based on difference in absorbance at 235 and 280 nm. Analyt. Biochem. 109
, 156–159.Google Scholar
Kalb, V. F. and Bernlohr, R. W. (1977). A new spectrophotometric assay for protein in cell extracts. Analyt. Biochem.
, 362–371.PubMedCrossRefGoogle Scholar
© Humana Press Inc., Totowa, NJ 2002