Advertisement

Detection of Proteins in Polyacrylamide Gels by Fluorescent Staining

  • Michael J. Dunn
Protocol
  • 122 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Techniques of polyacrylamide gel electrophoresis are often the method of choice for the analysis of patterns of gene expression in a wide variety of complex systems. In particular, two-dimensional polyacrylamide gel electrophoresis (2-DE) is the core technology for separating complex protein mixtures in the majority of proteome projects (1). This is due to its unrivalled power to separate simultaneously thousands of proteins and the availability of sophisticated computer software for the qualitative and quantitative analysis of differential patterns of protein expression (2). Once this analysis has indicated proteins of interest, it is now relatively straightforward to directly identify and characterize proteins from polyacrylamide gels using highly sensitive microchemical methods (3), particularly those based on mass spectrometry (4). This has resulted in gel electrophoresis becoming one of the most important methods of protein purification for subsequent identification and characterization.

Keywords

Silver Staining High Dynamic Range Dansyl Chloride Sophisticated Computer Software Complex Protein Mixture 
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.
    Dunn, M. J. and Görg, A. (2001) Two-dimensional polyacrylamide gel electrophoresis for proteome analysis, in Proteomics, From Protein Sequence to Function (Pennington, S. R. and Dunn, M. J., eds.), BIOS, Oxford, pp. 43–63.Google Scholar
  2. 2.
    Pleissner, K. P., Oswald, H., and Wegner, S. (2001) Image analysis of two-dimensional gels, in Proteomics, From Protein Sequence to Function (Pennington, S. R. and Dunn, M. J., eds.), BIOS, Oxford, pp. 131–149.Google Scholar
  3. 3.
    Wilkins, M. R. and Gooley, A. (1997) Protein identification in proteome analysis, in Proteome Research: New Frontiers in Functional Genomics (Wilkins, M. R., Williams, K. L., Appel, R. D. and Hochstrasser, D. F., eds.), Springer-Verlag, Berlin, pp. 35–64.Google Scholar
  4. 4.
    Patterson, S. D., Aebersold, R., and Goodlett, D. R. (2001) Mass spectrometry-based methods for protein identification and phosphorylation site analysis, in Proteomics, From Protein Sequence to Function (Pennington, S. R. and Dunn, M. J., eds.), BIOS, Oxford, pp. 87–130.Google Scholar
  5. 5.
    Neuhoff, V., Arold, N., Taube, D., and Erhardt, W. (1988) Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9, 255–262.PubMedCrossRefGoogle Scholar
  6. 6.
    Patton, W. F. (2001) Detecting proteins in polyacrylamide gels and on electroblot membranes, in Proteomics, From Protein Sequence to Function (Pennington, S. R. and Dunn, M. J., eds.), BIOS, Oxford, pp. 65–86.Google Scholar
  7. 7.
    Switzer, R. C., Merril, C. R., and Shifrin, S. (1979) A highly sensitive stain for detecting proteins and peptides in polyacrylamide gels. Analyt. Biochem. 98, 231–237.PubMedCrossRefGoogle Scholar
  8. 8.
    Rabilloud, T. (1990) Mechanisms of protein silver staining in polyacrylamide gels: A 10-year synthesis. Electrophoresis 11, 785–794.PubMedCrossRefGoogle Scholar
  9. 9.
    Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Analyt. Chem. 68, 850–858.CrossRefGoogle Scholar
  10. 10.
    Yan, J. X., Wait, R, Berkelman, T., Harry, R., Westbrook, J. A., Wheeler, C. H., Dunn, M. J. (2000) A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry. Electrophoresis 21, 3666–3672.PubMedCrossRefGoogle Scholar
  11. 11.
    Stephens, R. E. (1975) High-resolution preparative SDS-polyacrylamide gel electrophoresis: fluorescent visualization and electrophoretic elution-concentration of protein bands. Analyt. Biochem. 65, 369–379.PubMedCrossRefGoogle Scholar
  12. 12.
    Eng, P. R., and Parkes, C. O. (1974) SDS electrophoresis of fluorescamine-labeled proteins. Analyt. Biochem. 59, 323–325.PubMedCrossRefGoogle Scholar
  13. 13.
    Weidekamm, E., Wallach, D. F., and Fluckiger, R. (1973) A new sensitive, rapid fluorescence technique for the determination of proteins in gel electrophoresis and in solution. Analyt. Biochem. 5, 102–114.CrossRefGoogle Scholar
  14. 14.
    Barger, B. O., White, R. C., Pace, J. L., Kemper, D. L., Ragland, W. L. (1976) Estimation of molecular weight by polyacrylamide gel electrophoresis using heat stable fluors. Analyt. Biochem. 70, 327–335.PubMedCrossRefGoogle Scholar
  15. 15.
    O’Keefe, D. O. (1994) Quantitative electrophoretic analysis of proteins labeled with monobromobimane. Analyt. Biochem. 222, 86–94.PubMedCrossRefGoogle Scholar
  16. 16.
    Urwin, V. E. and Jackson, P. (1993) Two-dimensional polyacrylamide gel electrophoresis of proteins labeled with the fluorophore monobromobimane prior to first-dimensional isoelectric focusing: imaging of the fluorescent protein spot patterns using a charge-coupled device. Analyt. Biochem. 209, 57–62.PubMedCrossRefGoogle Scholar
  17. 17.
    Fey, S. J., Nawrocki, A., Larsen, M. R., Görg, A., Roepstorff, P., Skews, G. N., et al. (1997) Proteome analysis of Saccharomyces cerevisia: A methodological outline. Electrophoresis 18, 1361–1372.PubMedCrossRefGoogle Scholar
  18. 18.
    Ünlü, M., Morgan, M. E., and Minden, J. S. (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18, 2071–2077.PubMedCrossRefGoogle Scholar
  19. 19.
    Tonge, R., Shaw, J., Middleton, B., Rowlinson, R., Rayner, S., Young, J., et al. (2001) Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology. Proteomics 1, 377–396.PubMedCrossRefGoogle Scholar
  20. 20.
    Jackson, P., Urwin, V. E., and Mackay, C. D. (1988) Rapid imaging, using a cooled chargecoupled-device, of fluorescent two-dimensional polyacrylamide gels produced by labelling proteins in the first-dimensional isoelectric focusing gel with the fluorophore 2-methoxy-2,4-diphenyl-3(2H)furanone. Electrophoresis 9, 330–339.PubMedCrossRefGoogle Scholar
  21. 21.
    Herbert, B. R., Molloy, M. P., Gooley, A. A., Walsh, B. J., Bryson, W. G., and Williams, K. L. (1998) Improved protein solubility in 2-D electrophoresis using tributyl phopshine as the reducing agent. Electrophoresis 19, 845–851.PubMedCrossRefGoogle Scholar
  22. 22.
    Hartman, B. K. and Udenfriend, S. (1969) A method for immediate visualization of proteins in acrylamide gels and its use for preparation of antibodies to enzymes. Analyt. Biochem. 30, 391–394.PubMedCrossRefGoogle Scholar
  23. 23.
    Steinberg, T. H., Jones, L. J., Haugland, R. P., and Singer, V. L. (1996) SYPRO orange and SYPRO red protein gel stains: one-step fluorescent staining of denaturing gels for detection of nanogram levels of protein. Analyt. Biochem. 239, 223–237.PubMedCrossRefGoogle Scholar
  24. 24.
    Steinberg, T. H., Haugland, R. P., and Singer, V. L. (1996) Applications of SYPRO orange and SYPRO red protein gel stains. Analyt. Biochem. 239, 238–245.PubMedCrossRefGoogle Scholar
  25. 25.
    Steinberg, T. H., Lauber, W. M., Berggren, K., Kemper, C., Yue, S., and Patton, W. F. (1999) Fluorescence detection of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution Electrophoresis 20, 497–508.CrossRefGoogle Scholar
  26. 26.
    Steinberg, T., Chernokalskaya, E., Berggren, E., Lopez, M., Diwu, Z., Haugland, R., and Patton, W. (2000) Ultrasensitive fluorescence protein detection in isoelectric focusing gels using a ruthenium metal chelate stain. Electrophoresis 21, 486–496.PubMedCrossRefGoogle Scholar
  27. 27.
    Patton, W. F. (2002) A thousand points of light: the application of fluorescence detection technologies to two-dimensional gel electrophoresis and proteomics. Electrophoresis 21, 1123–1144.CrossRefGoogle Scholar
  28. 28.
    Yan, J. X., Harry, R. A., Spibey, C., and Dunn, M. J. (2000) Post-electrophoretic staining of proteins separated by two-dimensional gel electrophoresis using SYPRO dyes. Electro-phoresis 21, 3657–3665.CrossRefGoogle Scholar
  29. 29.
    Berggren, K., Steinberg, T. H., Lauber, W. M., Carroll, J. A., Lopez, M. F., Chernokalskaya, E., et al. (1999) A luminescent ruthenium complex for ultrasensitive detection of proteins immobilized on membrane supports. Analyt. Biochem. 276, 129–143.PubMedCrossRefGoogle Scholar
  30. 30.
    Berggren, K., Chernokalskaya, E., Steinberg, T. H., Kemper, C., Lopez, M. F., Diwu, Z., et al. (2000) Background-free, high sensitivity staining of proteins in one-and two-dimensional sodium dodecyl sulfate-polyacrylamide gels using a luminescent ruthenium complex. Electrophoresis. 21, 2509–2521.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Michael J. Dunn
    • 1
  1. 1.Department of NeuroscienceInstitute of PsychiatryLondonUK

Personalised recommendations