The Colorimetric Detection and Quantitation of Total Protein

Randall I. Krohn1

1 Pierce Biotechnology, Inc., Rockford, Illinois
Publication Name:  Current Protocols in Toxicology
Unit Number:  Appendix 3I
DOI:  10.1002/0471140856.txa03is23
Online Posting Date:  March, 2005
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Abstract

Protein quantification is an important step for handling protein samples for isolation and characterization; it is a prerequisite step before submitting proteins for chromatographic, electrophoretic, or immunochemical analysis and separation. Colorimetric methods are fast, simple, and not laborious. This unit describes a number of assays able to detect protein concentrations in the low microgram to milligram per milliliter ranges in a variety of formats.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: The Lowry Protein Assay for Determination of Total Proteins
  • Alternate Protocol 1: Modified Lowry Protein Assay for Determination of Total Proteins
  • Alternate Protocol 2: Microtiter Plate Modified Lowry Assay for Total Protein
  • Basic Protocol 2: The Bicinchoninic Acid (BCA) Assay for Determination of Total Protein
  • Alternate Protocol 3: Using Kits for BCA Measurements of Total Protein
  • Alternate Protocol 4: Microtiter Plate Assay for BCA Measurement of Total Protein
  • Basic Protocol 3: The Biuret Assay for Determining Total Protein
  • Alternate Protocol 5: Microtiter Plate Biuret Assay for Total Protein
  • Basic Protocol 4: The Coomassie Dye–Binding (Bradford) Assay for Determining Total Protein
  • Alternate Protocol 6: The Coomassie Plus Protein Assay for Determination of Total Protein
  • Alternate Protocol 7: Microtiter Plate Coomassie Assay for Total Protein
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: The Lowry Protein Assay for Determination of Total Proteins

  Materials
  • Standard protein: 2 mg/ml BSA (see recipe)
  • Sample buffer or solvent
  • Protein sample(s)
  • Lowry's Reagents C and D or D′ (see reciperecipes)

Alternate Protocol 1: Modified Lowry Protein Assay for Determination of Total Proteins

  • Modified Lowry Protein Assay Kit (Pierce) containing:
    • 2 mg/ml BSA in 0.9% (w/v) NaCl/0.05% (w/v) sodium azide
    • 2 N Folin‐Ciocalteu reagent: dilute fresh to 1 N
    • Modified Lowry's Reagent

Alternate Protocol 2: Microtiter Plate Modified Lowry Assay for Total Protein

  • Microtiter plate and cover or tape seals
  • 200‐µl multichannel pipettor
  • Microtiter plate reader for 750 nm

Basic Protocol 2: The Bicinchoninic Acid (BCA) Assay for Determination of Total Protein

  Materials
  • Protein standard: 2 mg/ml BSA (see recipe)
  • Sample buffer or solvent
  • Protein sample
  • BCA working reagent: mix 100 parts BCA reagent A with 2 parts reagent B (see reciperecipes for each reagent)

Alternate Protocol 3: Using Kits for BCA Measurements of Total Protein

  Additional Materials
  • BCA Protein Assay Reagent Kit (Pierce) or Bicinchoninic Acid Kit (Sigma) containing:
    • 2 mg/ml BSA in 0.9% NaCl/0.05% sodium azide (also see recipe)
    • BCA reagent A (also see recipe)
    • BCA reagent B (also see recipe)
  • 37°C water bath

Alternate Protocol 4: Microtiter Plate Assay for BCA Measurement of Total Protein

  • 96‐well microtiter plate with cover or tape seal
  • 200‐µl multichannel pipettor
  • Microtiter plate shaker
  • 37°C dry‐heat incubator
  • Microtiter plate reader

Basic Protocol 3: The Biuret Assay for Determining Total Protein

  Materials
  • Standard protein (also see )
  • Sample (unknown) protein
  • Biuret total protein reagent (Sigma Diagnostics; also see recipe)

Alternate Protocol 5: Microtiter Plate Biuret Assay for Total Protein

  • 96‐well microtiter plate and cover or tape sealer
  • 250‐µl multichannel pipettor and appropriate tips
  • Microtiter plate mixer
  • Microtiter plate reader

Basic Protocol 4: The Coomassie Dye–Binding (Bradford) Assay for Determining Total Protein

  Materials
  • Sample buffer or solvent
  • Protein standard (e.g., 2 mg/ml BSA; see recipe)
  • Protein sample
  • Coomassie dye reagent (Pierce or Bio‐Rad; also see recipe)

Alternate Protocol 6: The Coomassie Plus Protein Assay for Determination of Total Protein

  • Coomassie Plus Protein Assay Reagent Kit (Pierce) containing Coomassie Plus Protein Assay Reagent

Alternate Protocol 7: Microtiter Plate Coomassie Assay for Total Protein

  • Coomassie Plus Protein Assay Reagent Kit (Pierce) containing Coomassie Plus Protein Assay reagent
  • 96‐well microtiter plate
  • 300‐µl multichannel pipettor
  • Microtiter plate mixer
  • Microtiter plate reader
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Figures

  •   FigureFigure a0.3I.1 The reaction schematic for the Lowry Protein Assay.
  •   FigureFigure a0.3I.2 Graph of the color response curves obtained with Pierce's Modified Lowry Protein Assay Reagent using bovine serum albumin (BSA) and bovine gamma globulin (BGG). The standard tube protocol was performed and the color was measured at 750 nm in a Hitachi U‐2000 spectrophotometer.
  •   FigureFigure a0.3I.3 The reaction schematic for the BCA Protein Assay.
  •   FigureFigure a0.3I.4 Graph of the color response curves obtained with Pierce's BCA Protein Assay Reagent using bovine serum albumin (BSA) and bovine gamma globulin (BGG). The standard tube protocol was performed and the color was measured at 562 nm in a Hitachi U‐2000 spectrophotometer.
  •   FigureFigure a0.3I.5 The schematic of the biuret reaction.
  •   FigureFigure a0.3I.6 Graph of the color response curves obtained with Sigma's biuret Total Protein Reagent using bovine serum albumin (BSA) and bovine gamma globulin (BGG). The standard tube protocol was performed and the color was measured at 540 nm in a Hitachi U‐2000 spectrophotometer.
  •   FigureFigure a0.3I.7 The reaction schematic for the Coomassie Protein Assay.
  •   FigureFigure a0.3I.8 Graph of the color response curves obtained with Pierce's Coomassie Plus Protein Assay Reagent using bovine serum albumin (BSA) and bovine gamma globulin (BGG). The standard tube protocol was performed and the color was measured at 595 nm in a Hitachi U‐2000 spectrophotometer.

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Literature Cited

   Autenrieth, W. and Mink, F. 1915. Über kolorimetrische Bestimmungsmethoden:  Die quantitative Bestimmung von Harneiweiss. München Med. Wochenschr. 62:1417.
   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.
   Doumas, B.T., Bayse, D.D., and Carter, R.J. 1981. A candidate reference method for determination of total protein in serum. Clin. Chem. 27:1642.
   Fine, J. 1935. Biuret method of estimating albumin and globulin in serum and urine. Biochem. J. 29:799.
   Goa, J. 1953. A microbiuret method for protein determination. Scand. J. Clin. Lab. Invest. 5:218‐222.
   Gornall, A.G., Baardawill, C.J., and David, M.M. 1949. Determination of serum proteins by means of the biuret reagent. J. Biol. Chem. 177:751‐766.
   Hiller, A. 1926. Determination of albumin and globulin in urine. Proc. Soc. Exp. Biol. Med. 24:385.
   Kingsley, G.R. 1942. The direct biuret method for the determination of serum proteins as applied to photoelectric and visual colorimetry. J. Lab. Clin. Med. 27:840‐845.
   Legler, G., Muller‐Platz, C.M., Mentges‐Hettkamp, M., Pflieger, G., and Julich, E. 1985. On the chemical basis of the Lowry protein determination. Anal. Biochem. 150:278‐287.
   Lowry, O.H., Nira, J., Rosenbrough, A., Farr, L., and Randall, R.J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265‐275.
   Riegler, E. 1914. Eine kolorimetrische Bestimmungsmethode des Eiweisses. Z. Anal. Chem. 53:242‐254.
   Smith, P.K., Krohn, R.I., Hermanson, G.H., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goike, N.M., Olson, B.J., and Klenk, D.K. 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:76‐85.
   Weichselbaum, T.E. 1946. An accurate and rapid method for the determination of proteins in small amounts of blood serum and plasma. Am. J. Clin. Pathol. 16:40.
   Young, D.S. 1990. Effects of Drugs on Clinical Tests. (Suppl. 1 ,1991). AACC Press. Washington, D.C.
Key References
   Akins, R.E. and Tuan, R.S. 1992. Measurement of protein in 20 seconds using a microwave BCA assay. Biotechniques 12:496‐499.
  Use of the BCA protein assay in a microtiter‐plate format which utilizes a microwave oven as the heat source to shorten the color development time to 20  sec.
   Beyer, R.E. 1983. A rapid biuret assay for protein of whole fatty tissues. Anal. Biochem. 129:483‐485.
  Accurate total protein quantitation of fatty tissues was achieved by adding 0.1 ml 10% sodium deoxycholate, pH 8.0 and 2.9 ml biuret reagent to each protein pellet following an acetone/ether wash step. After sonication, each sample was heated for 30 sec in a boiling water bath to develop full color.
   Brown, A.J., Jarvis, K., and Hyland, K. 1989. Protein measurement using bicinchoninic acid: Elimination of interfering substances. Anal. Biochem. 180:136‐139.
  Describes a procedure using trichloroacetic acid and sodium deoxycholate to precipitate protein and thus remove soluble substances in the sample that would otherwise interfere in the BCA protein assay.
   Compton, S.J. and Jones, C.G. 1985. Mechanism of dye response and interference in the Bradford protein assay. Anal. Biochem. 151:369‐374.
  Found that the anionic form of the Coomassie dye reacts primarily with arginine residues within the macromolecular protein. Coomassie dye reacts to a lesser extent with other basic amino acid residues (His, Lys) and aromatic residues (Trp, Tyr, Phe) present in macromolecular proteins but not with the free amino acids. Dye binding is attributed to van der Waals forces and hydrophobic interactions. The interference seen with bases, detergents, and other compounds can be explained by their effects upon the equilibrium between the three dye forms (cationic, neutral, anionic).
   Crowley, L.V. 1969. Interference with certain chemical analyses caused by dextran. Am. J. Clin. Pathol. 51:425.
  Dextran at high concentrations causes a slight overestimation of the total protein concentration with the biuret reagent.
   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.
  A deoxycholate‐trichloroacetic acid protein precipitation technique that provides for rapid recovery of soluble and membrane‐bound proteins from interfering substances. Interference by lipids and nonionic or cationic detergents is alleviated by adding SDS.
   Peterson, G.L. 1979. Review of the Folin phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall. Anal. Biochem. 100:201‐220.
  A thorough review article that examines the reaction mechanism involved when protein reacts with the Lowry reagent. An extensive list of possibly interfering substances is presented along with methods of coping with those interfering substances. Finally, the method of Lowry is compared to other methods. There is an extensive list of references.
   Sorenson, K. and Brodbeck, U. 1986. A sensitive protein assay method using micro‐titer plates. Experientia 42:161‐162.
  A direct scale‐down of the BCA method for test tubes that is suitable for microtiter plates.
   Tal, M., Silberstein, A., and Nusser, D. 1980. Why does Coomassie Brilliant Blue interact differently with different proteins? J. Biol. Chem. 260:9976‐9980.
  Analysis of Scatchard plots showed that the number of Coomassie dye ligands bound to each protein is approximately proportional to the number of positive charges on the protein. About 1.5 to 3 dye molecules are bound to each positive charge on the protein.
   Watters, C. 1978. A one‐step biuret assay for protein in the presence of detergent. Anal. Biochem. 88:695‐698.
  A modified biuret reagent was formulated (sodium tartrate replaces sodium potassium tartrate, the sodium hydroxide concentration is reduced, and potassium iodide was deleted). When the modified biuret reagent was mixed with samples containing 2% detergent (SDS or sodium cholate or Triton X‐100), it resulted in less protein‐to‐protein variation among six proteins.
  Weichselbaum, 1946. See above.
  Used sodium potassium tartrate as a stabilizer and added potassium iodide to prevent autoreduction of the biuret reagent; however, this reagent was found to be unstable after long storage.
   Wiechelman, K., Braun, R., and Fitzpatrick, J. 1988. Investigation of the bicinchoninic acid protein assay: Identification of the groups responsible for color formation. Anal. Biochem. 175:231‐237.
  Cysteine, cystine, tryptophan, tyrosine, and the peptide bond are capable of reducing Cu2+ to Cu+, but the extent of color formation is not simply the sum of the contributions from the various color producing functional groups. At 60°C, tryptophan, tyrosine, and the peptide bond are more completely oxidized than they are at 37°C, which is observed by the much greater extent of color developed at the higher temperature.
Internet Resources
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  http://www.sigma-aldrich.com
  This site allows access to the Sigma Diagnostics product line and applications or other technical information on their products. Questions can be sent by e‐mail to the technical assistance department.
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