Quantification of Proteins by Staining in Polyacrylamide Gels

  • Bryan John Smith
Part of the Springer Protocols Handbooks book series (SPH)


Quantification of proteins is a common challenge. There are various methods described for estimation of the amount of protein present in a sample as described in Chapters 1–9. These quantify total protein present, but do not quantify one protein in a mixture of several. For this, the mixture must be resolved. Liquid chromatography achieves this, and the various proteins may be quantified by their absorbance at 220 or 280 nm. Microgram to submicrogram amounts of protein can be analyzed in this way, using microbore HPLC. Problems may occur if particular species chromatograph poorly (such as hydrophobic polypeptides on reverse-phase chromatography). An alternative is polyacrylamide gel electrophoresis (PAGE) as the mixture-resolving step, followed by protein staining and densitometry. Exceptions are small peptides that are not successfully resolved and stained in gels. Microgram to submicrogram amounts of protein (of over a few kilodaltons in size) may be quantified in this way. Although not every protein stain is best suited to this purpose, the method described herein is suitable for quantification of microgram-to-submicrogram amounts of proteins on sodium dodecyl sulfate (SDS) polyacrylamide gels (1,2).


Sodium Dodecyl Sulfate Zinc Salt Reactive Blue Standard Curve Equation Stain Protein Band 
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.


  1. 1.
    Harris, M. R. and Smith, B. J. (1983) A qualitative and quantitative study of subfractions of the histone H1° in various mammalian tissues. Biochem. J. 211, 763–766.PubMedGoogle Scholar
  2. 2.
    Smith, B. J., Toogood, C. I. A., and Johns, E. W. (1980) Quantitative staining of submicrogram amounts of histone and high-mobility group proteins on sodium dodecylsulphate-polyacrylamide gels. J. Chromatog. 200, 200–205.CrossRefGoogle Scholar
  3. 3.
    Neuhoff, V., Stamm, R., Pardowitz, I., Arold, N., Ehhardt, W., and Taube, D. (1990) Essential problems in quantification of proteins following colloidal staining with Coomassie Brilliant Blue dyes in polyacrylamide gels, and their solution. Electrophoresis 11, 101–117.PubMedCrossRefGoogle Scholar
  4. 4.
    Reisner, A. H., Nemes, P., and Bucholtz, C. (1975) The use of Coomassie brilliant blue G250 perchloric acid solution for staining in electrophoresis and isoelectric focusing on polyacrylamide gels. Anal. Biochem. 64, 509–516.PubMedCrossRefGoogle Scholar
  5. 5.
    Syrovy, I. and Hodny, Z. (1991) Staining and quantification of proteins separated by PAGE. J. Chromatog. 569, 175–196.CrossRefGoogle Scholar
  6. 6.
    O’Keefe, D. O. (1994) Quantitative electrophoretic analysis of proteins labeled with monobromobimane. Anal. Biochem. 222, 86–94.PubMedCrossRefGoogle Scholar
  7. 7.
    Ferreras, M., Gavilanes, J. G., and Garcia-Segura, J. M. (1993) A permanent Zn2+ reverse staining method for the detection and quantification of proteins in polyacrylamide gels. Anal. Biochem. 213, 206–212.PubMedCrossRefGoogle Scholar
  8. 8.
    Patton, W. F., Lam, L., Su, Q., Lui, M., Erdjument-Bromage, H., and Tempst, P. (1994) Metal chelates as reversible stains for detection of electroblotted proteins: application to protein microsequencing and immune-blotting. Anal. Biochem. 220, 324–335.PubMedCrossRefGoogle Scholar
  9. 9.
    Dráber, P. (1991) Quantification of proteins in sample buffer for sodium dodecyl sulfate-polyacrylamide gel electrophoresis using colloidal silver. Electrophoresis 12, 453–456.PubMedCrossRefGoogle Scholar
  10. 10.
    Henkel, A. W. and Bieger, S. C. (1994) Quantification of proteins dissolved in an electrophoresis sample buffer. Anal. Biochem. 223, 329–331.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1996

Authors and Affiliations

  • Bryan John Smith
    • 1
  1. 1.Celltech Therapeutics Ltd.SloughUK

Personalised recommendations