Spectrophotometric Determination of Protein Concentration

Michael H. Simonian1

1 Beckman Coulter Inc., Fullerton, California
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Appendix 3B
DOI:  10.1002/0471143030.cba03bs15
Online Posting Date:  August, 2002
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Abstract

This unit describes spectrophotometric and colorimetric methods for measuring the concentration of a sample protein in solution. Absorbance measured at 280 nm (A280) is used to calculate protein concentration by comparison with a standard curve or published absorptivity values for that protein (a280). Alternatively, absorbance measured at 205 nm (A205) is used to calculate the protein concentration. The A280 and A205 methods can be used to quantify total protein in crude lysates and purified or partially purified protein. A spectrofluorometer or a filter fluorometer can be used to measure the intrinsic fluorescence emission of a sample solution; this value is compared with the emissions from standard solutions to determine the sample concentration. The fluorescence emission method is used to quantify purified protein. This simple method is useful for dilute protein samples and can be completed in a short amount of time. There are two colorimetric methods: the Bradford colorimetric method, based upon binding of the dye Coomassie brilliant blue to the protein of interest, and the Lowry method, which measures colorimetric reaction of tyrosyl residues in the protein sample.

     
 
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Table of Contents

  • Basic Protocol 1: Using A280 to Determine Protein Concentration
  • Alternate Protocol 1: Using A205 to Determine Protein Concentration
  • Basic Protocol 2: Using Fluorescence Emission to Determine Protein Concentration
  • Reagents and Solutions
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1: Using A280 to Determine Protein Concentration

  Materials
  • 3 mg/ml standard protein solution (see recipe; optional)
  • Sample protein
  • Spectrophotometer with UV lamp

Alternate Protocol 1: Using A205 to Determine Protein Concentration

  • Brij 35 solution: 0.01% (v/v) Brij 35 (Sigma) in an aqueous solution appropriate for dissolving or diluting the sample protein

Basic Protocol 2: Using Fluorescence Emission to Determine Protein Concentration

  Materials
  • Standard protein solution prepared using the purified protein (see recipe)
  • Sample protein
  • Spectrofluorometer or filter fluorometer with an excitation cutoff filter ≤285 nm and an emission filter >320 nm
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Figures

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Literature Cited

Literature Cited
   Fasman, G.D. 1989. Practical Handbook of Biochemistry and Molecular Biology. CRC Press, Boca Raton, Fla.
   Freifelder, D. 1982. Physical Biochemistry: Applications to Biochemistry and Molecular Biology 2nd ed. W.H. Freeman, New York.
   Hawkins, B.K. and Honigs, D.E. 1987. A comparison of spectroscopic techniques for protein quantification in aqueous solutions. Am. Biotechnol. Lab. 5:26‐37.
   Layne, E. 1957. Spectrophotometric and turbidimetric methods for measuring proteins. Methods Enzymol. 3:447‐454.
   Pace, C.N., Vajdos, F., Fee, L., Grimsley, G., and Gray, T. 1995. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 4:2411‐2423.
   Scopes, R.K. 1974. Measurement of protein by spectrophotometry at 205 nm. Anal. Biochem. 59:277‐282.
   Stoscheck, C.M. 1990. Quantitation of protein. Methods Enzymol. 182:50‐68.
   Teale, F.W.J. 1960. The ultraviolet fluorescence of proteins in neutral solutions. Biochem. J. 76:381‐388.
   Warburg, O. and Christian, W. 1942. Isolierung und Kristallisation des Garungsferments Enolase. Biochem. Z. 310:384‐421.
Key References
   Chen, R.F. 1990. Fluorescence of proteins and peptides. In Practical Fluorescence, 2nd ed. (G.G. Guilbault, ed.) pp. 575‐682. Marcel Dekker, Inc., New York.
  Detailed discussion of intrinsic fluorescence of proteins and what factors affect fluorescence emission by the aromatic amino acids (see pp. 618‐663).
   Fasman, 1989. See above.
  Contains tables with absorptivities for UV spectrophotometric detection and tables with data on excitation and emission wavelengths for fluorescence detection of many proteins. Also includes a table with molecular weights for many characterized proteins.
   Stoscheck 1990. See above.
  Contains list of substances that can interfere with 205‐ and 280‐nm spectrophotometric measurements of proteins and of concentration limits for these substances.
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