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Spectrophotometric Determination of Protein Concentration

Gerald R. Grimsley1,  C. Nick Pace1

1The Texas A&M University System Health Science Center, College Station, Texas

Publication Name: 
Current Protocols in Protein Science
Unit Number: 
Unit 3.1
DOI: 
10.1002/0471140864.ps0301s33
Online Posting Date: 
November, 2004
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Learn more about the author(s): 
Gerald Grimsley
Learn more about the author(s): 
, Carlos Pace

Abstract

The concentration of a purified protein in solution is most conveniently and accurately measured using absorbance spectroscopy. The absorbance, A, is a linear function of the molar concentration, C, according to the Beer-Lambert law: A = × l × c, where e is the molar absorption coefficient and l is the cell path length. This unit provides protocols for calculation of for a folded or unfolded protein, making use of the average values for the three contributing chromophores in proteins (the side chains of Trp, Tyr, and Cys). A basic protocol describes how to measure the concentration of a protein using the calculated and the Beer-Lambert law. A sensitive method is provided for measuring the concentration of proteins that contain few if any tryptophan or tyrosine residues, and a simple method is provided for estimating total protein concentration in crude extracts.

Keywords: protein concentration; molar absorption coefficient; molar extinction coefficient; near UV absorbance; tryptophan absorbance; tyrosine absorbance

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Calculation of the Molar Absorption Coefficient () of a Protein
  • Basic Protocol 2: Determination of the Molar Absorption Coefficient () for a Folded Protein
  • Basic Protocol 3: Determination of Protein Concentration by Absorbance Spectroscopy Using the Molar Absorption Coefficient
  • Basic Protocol 4: Determination of Protein Concentration by Absorption Spectroscopy at 205 nm
  • Basic Protocol 5: Spectrophotometric Determination of Total Protein Concentration in Crude Protein Extracts
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 2: Determination of the Molar Absorption Coefficient () for a Folded Protein

 Materials
  • 8 M nitric acid
  • Ethanol
  • 20 mM potassium phosphate buffer, pH 6.5 (appendix 2E)
  • Protein stock solution: ~1 mg protein in 0.25 ml of 20 mM potassium phosphate buffer, pH 6.5 (see appendix 2E for buffer)
  • 6.6 M guanidine·HCl (appendix 3A) in 40 mM potassium phosphate buffer, pH 6.5 (appendix 2E); also see Critical Parameters
  • Double-beam absorbance spectrophotometer
  • Two quartz cuvettes, 10 mm long × 4 mm wide

Basic Protocol 3: Determination of Protein Concentration by Absorbance Spectroscopy Using the Molar Absorption Coefficient

 Materials
  • Solution of pure protein in either distilled water or buffer
  • Spectrophotometer with UV and visible lamps
  • Quartz cuvette

Basic Protocol 4: Determination of Protein Concentration by Absorption Spectroscopy at 205 nm

 Materials
  • Protein sample
  • Brij 35 solution: 0.01% (v/v) Brij 35 (Sigma) in an aqueous solution appropriate for dissolving the sample protein
  • Spectrophotometer with UV and visible lamps
  • Quartz cuvette

Basic Protocol 5: Spectrophotometric Determination of Total Protein Concentration in Crude Protein Extracts

 Materials
  • Sample of protein extract
  • Spectrophotometer with UV and visible lamps
  • Quartz cuvette
Looking for materials?  Suppliers Guide
     
 
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Literature Cited

Literature Cited
    Bradford, M.M. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.
    Edelhoch, H. 1967. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6:1948-1954.
    Gill, S.C. and von Hippel, P.H. 1989. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182:319-326.
    Leach, S.J. and Scheraga, H.A. 1960. Effect of light scattering on ultraviolet difference spectra. J. Am. Chem. Soc. 82:4790-4792.
    Lowry, O.H., Rosebrough, J.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. and Schmid, F.X. 1997. How to determine the molar absorbance coefficient of a protein. In Protein Structure: A Practical Approach. (T.E. Creighton, ed.) pp. 253-259. IRL Press, Oxford, U.K.
    Pace, C.N. and Scholtz, J.M. 1997. Measuring the conformational stability of a protein. In Protein Structure: A Practical Approach (T.E. Creighton, ed.) pp. 299-321. IRL Press, Oxford, U.K.
    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. Prot. Sci. 4:2411-2423.
    Scopes, R.K. 1974. Measurement of protein by spectrophotometry at 205 nm. Anal. Biochem. 59:277-282.
    Schmid, F.X. 1997. Optical spectroscopy to characterize protein conformation and conformational changes. In Protein Structure: A Practical Approach. (T.E. Creighton, ed.) pp. 261-297. IRL Press, Oxford, U.K.
    Stoscheck, C.M. 1990. Quantitation of protein. Methods Enzymol. 182:50-68.
    Warburg, O. and Christian, W. 1942. Isolierung und Kristallisation des Garungsferments Enolase. Biochem. Z. 310:384-421.
     
 
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Author Notes

Carlos Pace
June 10, 2009

The protocol discussed in this article is extremely useful and still important today (5 years later, June 2009).  Much in the protocol is based on the primary research findings published in our paper below: 

Title: HOW TO MEASURE AND PREDICT THE MOLAR ABSORPTION-COEFFICIENT OF A PROTEIN
Author(s): PACE, CN; VAJDOS, F; FEE, L, et al.
Source: PROTEIN SCIENCE   Volume: 4   Issue: 11   Pages: 2411-2423   Published: NOV 1995
Times Cited: 1,447

As of June 2009 this is the MOST CITED paper in PROTEIN SCIENCE.

Nick Pace

Gerald Grimsley
June 12, 2009

Based upon the number of requests for reprints we have received over the years, and the fact that the journal article upon which this protocol is taken is the most cited paper in Protein Science (to date), I would say our method given here is still very useful to determine protein concentration.  All one needs is a spectrophotometer, reasonably pure protein, and know the number of Trp, Tyr and S-S bonds that the protein contains.

Gerald Grimsley

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vbc
Posted on October 20, 2009 - 8:08am

This is described in the first basic protocol of the unit, but you can also get some information on this by looking at the Pace et al. references listed under Literature Cited

Esere Akpatason (not verified)
Posted on October 20, 2009 - 5:38am

how to determine the molar absortion coefficient of a protien

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