Assays for Determination of Protein Concentration

Bradley J.S.C. Olson1, John Markwell2

1 Michigan State University, East Lansing, Michigan, 2 University of Nebraska, Lincoln, Nebraska
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 3.4
DOI:  10.1002/0471140864.ps0304s48
Online Posting Date:  May, 2007
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Abstract

Biochemical analysis of proteins relies on accurate quantitation of protein concentration. This unit describes how to perform commonly used protein assays, e.g., Lowry, Bradford, BCA, and UV spectroscopic protein assays. The primary focus of the unit is assay selection, emphasizing sample and buffer compatibility. Protein assay standard curves and data processing fundamentals are discussed in detail. This unit also details high-throughput adaptations of the commonly used protein assays, and also contains a protocol for BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels, which is reliable, inexpensive, and quick.

Keywords: protein assay; spectrophotometry; SDS-PAGE

     
 
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Table of Contents

  • Unit Introduction
  • Strategic Planning
  • Basic Protocol 1: The Lowry Assay
  • Basic Protocol 2: The Bradford Assay
  • Alternate Protocol 1: Microtiter Plate Bradford
  • Basic Protocol 3: BCA Assay
  • Alternate Protocol 2: Microtiter Plate BCA Assay
  • Basic Protocol 4: UV Absorbance to Measure Protein Concentration
  • Support Protocol 1: Standard Curves and Data Processing
  • Support Protocol 2: Acetone Precipitation of Protein
  • Support Protocol 3: TCA Precipitation of Protein
  • Alternate Protocol 3: Alkylation of Reductant and BCA Assay of Protein Samples in SDS-Page Loading Buffer
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: The Lowry Assay

 Materials
  • 1 mg/ml protein standard (e.g., BSA, albumin, or -globulin; see Support Protocol 1)
  • Sample
  • Lowry assay mix (see recipe)
  • Freshly prepared diluted Folin-Ciocalteu reagent (see recipe)
  • Test tubes (e.g., 16 × 125–mm)
  • Spectrophotometer warmed up and set to 660 nm (or other appropriate wavelength) and cuvette

Basic Protocol 2: The Bradford Assay

 Materials
  • 1 mg/ml protein standard (e.g., BSA, albumin, or -globulin; see Support Protocol 1)
  • Protein sample
  • 1 M NaOH (optional)
  • Bradford reagent (see recipe)
  • Spectrophotometer warmed up at least 15 min before use and set to 595 nm, and cuvette
  • Test tubes (e.g., 10 × 75–mm for the micro assay or 17 × 100–mm for the macro assay)

Alternate Protocol 1: Microtiter Plate Bradford

 Materials
  • Sample
  • 1 mg/ml protein standard (e.g., BSA; see Support Protocol 1)
  • Bradford reagent (see recipe)
  • 1 M NaOH
  • Microtiter plate (see Strategic Planning)
  • Multichannel pipettor or repeating pipettor, optional
  • Microtiter plate reader (see Strategic Planning)

Basic Protocol 3: BCA Assay

 Materials
  • Macro-BCA or micro-BCA assay solution (see recipes)
  • Samples
  • 1 mg/ml standard proteins (e.g., BSA, albumin, or -globulin; see Support Protocol 1)
  • Test tubes (e.g., 10 × 75–mm for micro-BCA assay or 13 × 100–mm for macro-BCA assay)
  • Spectrophotometer warmed up and set to 562 nm, and cuvette
  • 60°C (or 37°C) heating block or water bath

Alternate Protocol 2: Microtiter Plate BCA Assay

 Materials
  • Micro-BCA working solution (see recipe)
  • 1 mg/ml protein standard (e.g., BSA; see Support Protocol 1)
  • Microtiter plate(s) (BD Falcon 353915; Costar 3370; Whatman 7701-1350)
  • Microtiter plate reader, warmed up and ready to read at 562 nm
  • 60°C (or 37°C) oven or water bath

Basic Protocol 4: UV Absorbance to Measure Protein Concentration

 Materials
  • Sample
  • Protein standard prepared in the same buffer as unknown
  • Sample buffer
  • Spectrophotometer and quartz or UV-transparent cuvettes

Support Protocol 2: Acetone Precipitation of Protein

 Materials
  • 100% acetone, –20°C
  • Protein sample
  • Protein assay–compatible buffer
  • –20°C freezer
  • 1.5-ml microcentrifuge tubes (acetone-compatible, not polycarbonate)
  • Microcentrifuge

Support Protocol 3: TCA Precipitation of Protein

 Materials
  • 80% (w/v) TCA
  • Protein sample
  • 80% acetone, –20°C
  • Protein assay–compatible buffer
  • –20°C freezer
  • 1.5-ml centrifuge tubes (acetone-compatible, not polycarbonate)
  • Microcentrifuge

Alternate Protocol 3: Alkylation of Reductant and BCA Assay of Protein Samples in SDS-Page Loading Buffer

 Materials
  • Stock protein standards
  • 6× SDS-PAGE loading dye without bromophenol blue (unit 10.1; see recipe)
  • Unknowns
  • 20 mM iodoacetamide solution (see recipe)
  • Additional reagents and equipment for BCA assay (see Basic Protocol 3)
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Figures

  •  FigureFigure 3.4.1 Flow chart for selecting a protein assay. To use the chart, begin with step A to find the protein assays that are most compatible with the sample. Then proceed to step B to obtain the list of assays compatible with the buffer system. Compare the results from each step to find the most compatible assay for the sample. Refer to the text for the assay and Table 3.4.1 to confirm assay compatibility before using a protein assay strategy. Abbreviations: 2-ME, 2-mercaptoethanol; DTT, dithiothreitol; SDS, sodium dodecyl sulfate.
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  •  FigureFigure 3.4.2 A sample Lowry protein assay standard curve produced using BSA at triplicate points of 0, 10, 20, 30, 40, and 50 µg. The data are fit with a linear regression by the line y = 153.06x + 0.179 with an R2 value of 0.992. The data table used to generate the figure and depiction of a typical Lowry assay is shown in Table 3.4.2.
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  •  FigureFigure 3.4.3 Absorbance spectra of the Bradford reagent and the Bradford reagent bound to 20 µg of BSA standard. The free reagent (solid line) has an absorbance peak of 470 nm whereas the Bradford reagent complexed with protein (dashed line) has an absorbance peak near 600 nm. Note that the unbound dye partially overlaps with the bound form of the reagent and thus leads to the nonlinear response of the Bradford assay.
    View Image
  •  FigureFigure 3.4.4 A sample micro-Bradford assay standard curve. BSA standard was added in triplicate at 0, 0.25, 1.25, 2.5, 5, 7.5, and 10 µg. Note that the assay does not respond linearly to the concentration of standard. The data is fit with the equation y = 4.5898x2 + 14.424x – 10.694 and has an R2 value of 0.9979. The data table used to generate the figure and depiction of a typical micro-Bradford assay is shown in Table 3.4.3.
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  •  FigureFigure 3.4.5 A sample micro-BCA assay standard curve with triplicate BSA standard points of 0, 0.625, 3.125, 6.25, 12.5, and 18.75 µg. Note that the curve is linear across a wide range and is fit with the equation y = 10.571x – 2.0254 and has a R2 value of 0.9893. The data table used to generate the figure and depiction of a typical micro-BCA assay is shown in Table 3.4.4.
    View Image
  •  FigureFigure 3.4.6 Data for a sample Lowry protein assay standard curve produced using BSA standard at triplicate points of 0, 10, 20, 30, 40, and 50 µg. The data are identical to those in Figure 3.4.2 except that BSA (µg) is plotted on the x axis and A660 is plotted on the y axis. The data are fit with a linear regression by the line y = 0.0065x + 0.0001 with an R2 value of 0.992. Note that to use the equation to return protein concentration, the equation must be rearranged.
    View Image

Videos

Literature Cited

Literature Cited
    Ballentine, R. 1957. Determination of total nitrogen and ammonia. Methods Enzymol. 3:984-995.
    Bensadoun, A. and Weinstein, D. 1976. Assay of proteins in the presence of interfering materials. Anal. Biochem. 70:241-250.
    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.
    Dulley, J.R. and Grieve, P.A. 1975. A simple technique for eliminating interference by detergents in the Lowry method of protein determination. Anal. Biochem. 64:136-141.
    Fasman, G.D. 1989. Practical Handbook of Biochemistry and Molecular Biology. CRC Press, Boca Raton, Florida.
    Gill, S.C. and von Hippel, P.H. 1989. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182:319-326.
    Gornall, A.G., Bardawill, C.S., and David, M.M. 1949. Determination of serum proteins by means of the biuret method. J. Biol. Chem. 177:751-766.
    Hartree, E.E. 1972. Determination of protein: A modification of the Lowry method that gives a linear photometric response. Anal. Biochem. 48:422-427.
    Hill, H.D. and Straka, J.G. 1988. Protein determination using bicinchoninic acid in the presence of sulfhydryl reagents. Anal. Biochem. 170:203-208.
    Layne, E. 1957. Spectrophotometric and turbidimetric methods for measuring proteins. Methods Enzymol. 3:447-455.
    Lovrien, R. and Matulis, D. 1995. Assays for total protein. Curr. Protoc. Protein Sci. 3.4.4-3.4.24.
    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.
    Mandula, H., Parepally, J.M., Feng, R., and Smith, O.R. 2006. Role of site-specific binding to plasma albumin in drug availability to brain. J. Pharmacol. Exp. Ther. 317:667-675.
    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.
    Stoscheck, C.M. 1990. Quantitation of protein. Methods Enzymol. 182:50-68.
    Wiechelman, K.J., Braun, R.D., and Fitzpatrick, J.D. 1988. Investigation of the bicinchoninic acid protein assay: Identification of the groups responsible for color formation. Anal. Biochem. 175:231-237.
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