Spectrophotometric and Colorimetric Determination of Protein Concentration

Michael H. Simonian1, John A. Smith2

1 Beckman Coulter, Inc., Fullerton, California, 2 University of Alabama at Birmingham, Birmingham, Alabama
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 10.1A
DOI:  10.1002/0471142727.mb1001as76
Online Posting Date:  November, 2006
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Abstract

This unit describes spectrophotometric and colorimetric methods for measuring the concentration of a sample protein in solution. Absorbance measurement at 280 nm is used to calculate protein concentration by comparison with a standard curve or published absorptivity values for that protein. An alternate protocol uses absorbance at 205 nm to calculate the protein concentration. Both methods can be used to quantitate total protein in crude lysates and purified or partially purified protein. Use of a spectrofluorometer or a filter fluorometer to measure the intrinsic fluorescence emission of a sample solution is also described. The measurement is compared with the emissions from standard solutions to determine the concentration of purified protein. The Bradford colorimetric method, based upon binding of the dye Coomassie brilliant blue to an unknown protein, is also presented, as is the Lowry method, which measures colorimetric reaction of tyrosyl residues in an unknown.

     
 
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Table of Contents

  • Section I: Quantitation of Proteins
  • 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
  • Basic Protocol 3: Using the Bradford Method to Determine Protein Concentration
  • Alternate Protocol 2: Using the Lowry Method to Determine Protein Concentration
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Using A280 to Determine Protein Concentration

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

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
  • recipeSpectrophotometric protein standard solution (see recipe) prepared using the purified protein
  • Sample protein
  • Spectrofluorometer or filter fluorometer with an excitation cutoff filter at ≤285 nm and an emission filter at >320 nm
  • Quartz fluorometer cuvette

Basic Protocol 3: Using the Bradford Method to Determine Protein Concentration

  Materials
  • recipeColorimetric standard protein solution (0.5 mg/ml BSA; see recipe)
  • 0.15 M NaCl
  • recipeCoomassie brilliant blue solution (see recipe)
  • 1 ml, 1‐cm‐path‐length microcuvette

Alternate Protocol 2: Using the Lowry Method to Determine Protein Concentration

  • 0.15% (w/v) sodium deoxycholate
  • 72% (w/v) trichloroacetic acid (TCA)
  • recipeCopper tartrate/carbonate (CTC) solution (see recipe)
  • 20% (v/v) Folin‐Ciocalteu reagent
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Figures

Videos

Literature Cited

   Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72:248‐254.
   Brogdon, W.G. and Dickinson, C.M. 1983. A microassay system for measuring esterase activity and protein concentration in small samples and in high‐pressure liquid chromatography eluate fractions. Anal. Biochem. 131:499‐503.
   Cadman, E., Bostwick, J.R., and Eichberg, J. 1979. Determination of protein by a modified Lowry procedure in the presence of some commonly used detergents. Anal. Biochem. 96:21‐23.
   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.
   Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265‐275.
   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.
   Peterson, G.L. 1977. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 83:346‐356.
   Peterson, G.L. 1979. Review of the Folin phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall. Anal. Biochem. 100:201‐220.
   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).
   Darbre, A. 1986. Analytical methods. In Practical Protein Chemistry: A Handbook (A. Darbre, ed.) pp. 227‐335. John Wiley & Sons, New York.
  Describes colorimetric methods for protein determination including the Lowry method and several modifications, the biuret method and several modifications, and the Bradford method (see pp. 284‐295).
   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 and colorimetric measurements of proteins and of concentration limits for these substances.
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