Assays for Total Protein

Rex Lovrien1, Daumantas Matulis2

1 University of Minnesota, St. Paul, Minnesota, 2 Institute of Biotechnology, Vilnius, Lithuania
Publication Name:  Current Protocols in Microbiology
Unit Number:  Appendix 3A
DOI:  10.1002/9780471729259.mca03as00
Online Posting Date:  October, 2005
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Abstract

This unit describes three copper‐based assays to quantitate total protein: the biuret method, a variation of the Lowry method (Hartree‐Lowry method), and the bicinchoninic acid (BCA) assay. Acid hydrolysis of a protein is coupled with ninhydrin detection to quantitate amino acid content of a sample. Ultraviolet spectrophotometry is used to measure total protein and evaluate samples for the presence of contaminants. The Coomassie dye binding, or Bradford, assay is a quite simple assay and frequently is quite sensitive, although it sometimes gives a variable response depending on how well or how poorly the protein binds the dye in acid pH. Finally, dry weight measurement is used to quantitate pure protein. Support protocols describe heat sealing glass tubes for acid hydrolysis, sample dialysis in polyacrylamide gel wells to remove low‐molecular‐weight contaminants, and TCA precipitation to precipitate and concentrate proteins and remove low‐molecular‐weight contaminants.

     
 
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Table of Contents

  • Basic Protocol 1: Hartree‐Lowry Assay for Quantitation of Total Protein
  • Basic Protocol 2: Bicinchoninic Acid (BCA) Assay for Quantitation of Total Protein
  • Basic Protocol 3: Ultraviolet Absorption to Measure Total Protein
  • Basic Protocol 4: Coomassie Dye–Binding Assay (Bradford Assay) to Measure Total Protein
  • Basic Protocol 5: Trichloroacetic Acid Precipitation of Protein Samples
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Hartree‐Lowry Assay for Quantitation of Total Protein

  Materials
  • Calibration standard: 300 µg/ml crystalline BSA in water
  • Buffer or solvent used to prepare the protein‐containing sample
  • Sample containing protein at 100 to 600 µg/ml
  • Hartree‐Lowry reagents A, B, and C (see reciperecipes)
  • 50°C water bath
  • Spectrophotometer and 1‐cm cuvettes

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

  Materials
  • Calibration standard: 1 mg/ml BSA
  • Buffer or solvent used to prepare the protein‐containing sample
  • Sample containing protein
  • BCA reagent A/reagent B mix (see recipe)
  • Spectrophotometer and cuvettes

Basic Protocol 3: Ultraviolet Absorption to Measure Total Protein

  Materials
  • Protein sample, pH <8
  • Spectrophotometer and fused‐silica or quartz cuvettes

Basic Protocol 4: Coomassie Dye–Binding Assay (Bradford Assay) to Measure Total Protein

  Materials
  • Calibration standards: 1.5 mg/ml BSA and 1.5 mg/ml lysozyme
  • Buffer or solvent used to prepare the protein‐containing sample
  • Sample containing protein
  • Coomassie dye reagent (see recipe) or commercial Coomassie reagent (Bio‐Rad, Pierce)
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Figures

Videos

Literature Cited

Literature Cited
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   Bencze, W.L. and Schmid, K. 1957. Determination of tyrosine and tryptophan in proteins. Anal. Chem. 29:1193‐1196.
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   Beyer, R.E. 1983. A rapid biuret assay for protein of whole fatty tissues. Anal. Biochem. 129:483‐485.
   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.
   Chang, Y.‐C. 1992. Efficient precipitation and accurate quantitation of detergent‐solubilized membrane proteins. Anal. Biochem. 205:22‐26.
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   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.
   Peterson, G.L. 1983. Determination of total protein. Methods Enzymol. 91:95‐119.
   Pierce, J. and Suelter, C.H. 1977. An evaluation of the Coomassie brilliant blue G‐250 dye‐binding method for quantitative protein determination. Anal. Biochem. 81:478‐480.
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   Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., and Klenk, D.C. 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:76‐85.
   Sober, H.A. (ed.) 1970. Molar extinction coefficients and E1% values for proteins at selected wavelengths of the ultraviolet and visible region. In Handbook of Biochemistry, Selected Data for Molecular Biology, pp. C71‐C98. Chemical Rubber Co. Press, Cleveland.
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   Van Kley, H. and Hale, S.M. 1977. Assay for protein by dye binding. Anal. Biochem. 81:485‐487.
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