Advertisement

The Bradford Method For Protein Quantitation

  • Nicholas J. Kruger
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

A rapid and accurate method for the estimation of protein concentration is essential in various areas of biology and biochemistry. An assay originally described by Bradford (1) has become the preferred method for quantifying protein in many laboratories. This technique is simpler, faster, and more sensitive than the Lowry method. Moreover, when compared with the Lowry method, it is subject to less interference by common reagents and non-protein components of biological samples (see Note 1). Despite the introduction of alternative protein assays, the Bradford method remains a popular technique, with the original article (1) being cited over 3,500 times in primary research papers in 2006, thirty years after its initial publication.

The Bradford assay relies on the binding of the dye Coomassie Blue G250 to protein. Detailed studies indicate that the free dye can exist in four different ionic forms for which the pKa values are 1.15, 1.82 and 12.4 (2). Of the three charged forms of the dye that predominate in the acidic assay reagent solution, the more cationic red and green forms have absorbance maxima at 470 nm and 650 nm, respectively. In contrast, the more anionic blue form of the dye, which binds to protein, has an absorbance maximum at 590 nm. Thus, the quantity of protein can be estimated by determining the amount of dye in the blue ionic form. This is usually achieved by measuring the absorbance of the solution at 595 nm (see Note 2).

Keywords

Absorbance Maximum Bradford Assay Reagent Blank Lowry Method Guanidine Hydrochloride 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    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.PubMedCrossRefGoogle Scholar
  2. 2.
    Chial, H.J., Thompson, H.B., and Splittgerber, A.G. (1993) A spectral study of the charge forms of Coomassie Blue G. Anal. Biochem. 209, 258–266.PubMedCrossRefGoogle Scholar
  3. 3.
    Compton, S.J. and Jones, C.G. (1985) Mechanism of dye response and interference in the Bradford protein assay. Anal. Biochem. 151, 369–374.PubMedCrossRefGoogle Scholar
  4. 4.
    Congdon, R.W., Muth, G.W., and Splittgerber, A.G. (1993) The binding interaction of Coomassie Blue with proteins. Anal. Biochem. 213, 407–413.PubMedCrossRefGoogle Scholar
  5. 5.
    Friendenauer, S. and Berlet, H.H. (1989) Sensitivity and variability of the Bradford protein assay in the presence of detergents. Anal. Biochem. 178, 263–268.CrossRefGoogle Scholar
  6. 6.
    Reade, S.M. and Northcote, D.H. (1981) Minimization of variation in the response to different proteins of the Coomassie Blue G dye-binding assay for protein. Anal. Biochem. 116, 53–64.CrossRefGoogle Scholar
  7. 7.
    Stoscheck, C.M. (1990) Increased uniformity in the response of the Coomassie Blue protein assay to different proteins. Anal. Biochem. 184, 111–116.PubMedCrossRefGoogle Scholar
  8. 8.
    Spector, T. (1978) Refinement of the Coomassie Blue method of protein quantitation. A simple and linear spectrophotometric assay for <0.5 to 50 g of protein. Anal. Biochem. 86, 142–146.PubMedCrossRefGoogle Scholar
  9. 9.
    Pande, S.V. and Murthy, M.S.R. (1994) A modified micro-Bradford procedure for elimination of interference from sodium dodecyl sulfate, other detergents, and lipids. Anal. Biochem. 220, 424–426.PubMedCrossRefGoogle Scholar
  10. 10.
    Zuo, S.-S. and Lundahl, P. (2000) A micro-Bradford membrane protein assay. Anal. Biochem. 284, 162–164.PubMedCrossRefGoogle Scholar
  11. 11.
    Whiffen, L.K., Midgley, D.J., and McGee, P.A. (2007) Polyphenolic compounds interfere with quantification of protein in soil extracts using the Bradford method. Soil Biol. Biochem. 39, 691–694.CrossRefGoogle Scholar
  12. 12.
    Sedmak, J.J. and Grossberg, S.E. (1977) A rapid, sensitive and versatile assay for protein using Coomassie Brilliant Blue G250. Anal. Biochem. 79, 544–552.PubMedCrossRefGoogle Scholar
  13. 13.
    Peterson, G.L. (1983) Coomassie Blue Dye Binding Protein Quantitation Method, in Methods in Enzymology, vol. 91 (Hirs, C.H.W. and Timasheff, S.N., eds.) Academic, New York.Google Scholar
  14. 14.
    Kirazov, L.P., Venkov, L.G., and Kirazov, E.P. (1993) Comparison of the Lowry and the Bradford protein assays as applied for protein estimation of membrane-containing fractions. Anal. Biochem. 208, 44–48.PubMedCrossRefGoogle Scholar
  15. 15.
    Wilson, C.M. (1979) Studies and critique of Amido Black 10B, Coomassie Blue R and Fast Green FCF as stains for proteins after polyacrylamide gel electrophoresis. Anal. Biochem. 96, 263–278.PubMedCrossRefGoogle Scholar
  16. 16.
    Zor, T. and Selinger, Z. (1996) Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies. Anal. Biochem. 236, 302–308.PubMedCrossRefGoogle Scholar
  17. 17.
    Redinbaugh, M.G. and Campbell, W.H. (1985) Adaptation of the dye-binding protein assay to microtiter plates. Anal. Biochem. 147, 144–147.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Nicholas J. Kruger
    • 1
  1. 1.Department of Plant SciencesUniversity of OxfordOxfordUK

Personalised recommendations