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Background-Free Protein Detection in Polyacrylamide Gels and on Electroblots Using Transition Metal Chelate Stains

  • Wayne F. Patton
Protocol
  • 91 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Electrophoretically separated proteins may be visualized using organic dyes such as Ponceau Red, Amido Black, Fast Green, or most commonly Coomassie Brilliant Blue (1,2). Alternatively, sensitive detection methods have been devised using metal ions and colloids of gold, silver, copper, carbon, or iron (3-12). Metal chelates form a third class of stains, consisting of transition metal complexes that bind avidly to proteins resolved in polyacrylamide gels or immobilized on solid-phase membrane supports (13-27). In recent years, metal chelate stains have been designed and optimized specifically for compatibility with commonly used microchemical characterization procedures employed in proteomics. The metal chelate stains are simple to implement, and do not contain extraneous chemicals such as glutaraldehyde, formaldehyde, or Tween-20 that are well known to interfere with many downstream protein characterization procedures.

Keywords

Linear Dynamic Range Colorimetric Detection Protein Blot Luminescent Detection Lectin Blotting 
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.
    Merril, C. (1987) Detection of proteins separated by electrophoresis, in Advances in Electrophoresis, Vol. 1 (Chrambach, A., Dunn, M., and Radola, B., eds.), VCH Press, Germany/Switzerland/Great Britian/New York, pp. 111–139.Google Scholar
  2. 2.
    Wirth, P. and Romano, A. (1995) Staining methods in gel electrophoresis, including the use of multiple detection methods. J. Chromatogr. A, 698, 123–143.PubMedCrossRefGoogle Scholar
  3. 3.
    Hancock, K. and Tsang, V. (1983) India ink staining of proteins on nitrocellulose paper. Analyt. Biochem. 133, 157–162.PubMedCrossRefGoogle Scholar
  4. 4.
    Moeremans, M., Daneels, G., and De Mey, J. (1985) Sensitive colloidal metal (gold or silver) staining of protein blots on nitrocellulose membranes. Analyt. Biochem. 145, 315–321.PubMedCrossRefGoogle Scholar
  5. 5.
    Moeremans, M., Raeymaeker, M., Daneels, G., and De Mey, J. (1986) Ferridye: colloidal iron binding followed by Perls’ reaction for the staining of proteins transferred from sodium dodecyl sulfate gels to nitrocellulose and positively charged nylon membranes. Analyt. Biochem. 153, 18–22.PubMedCrossRefGoogle Scholar
  6. 6.
    Hunter, J. and Hunter, S. (1987) Quantification of proteins in the low nanogram range by staining with the colloidal gold stain Aurodye,. Analyt. Biochem. 164, 430–433.PubMedCrossRefGoogle Scholar
  7. 7.
    Egger, D. and Bienz, K. (1987) Colloidal gold and immunoprobing of proteins on the same nitrocellulose blot. Analyt. Biochem. 166, 413–417.PubMedCrossRefGoogle Scholar
  8. 8.
    Yamaguchi, K. and Asakawa, H. (1988) Preparation of colloidal gold for staining proteins electrotransferred onto nitrocellulose membranes. Analyt. Biochem. 172, 104–107.PubMedCrossRefGoogle Scholar
  9. 9.
    Li, K., Geraerts, W., van Elk, R., and Joosse, J. (1988) Fixation increases sensitivity of india ink staining of proteins and peptides on nitrocellulose paper. Analyt. Biochem. 174, 97–100.PubMedCrossRefGoogle Scholar
  10. 10.
    Li, K., Geraerts, R., van Elk, R., and Joosse, J. (1989) Quantification of proteins in the subnanogram and nanogram range: comparison of the Aurodye, Ferridye, and India Ink staining methods. Analyt. Biochem. 182, 44–47.PubMedCrossRefGoogle Scholar
  11. 11.
    Root, D. and yReisler, E. (1989) Copper iodide staining of protein blots on nitrocellulose membranes. Analyt. Biochem. 181, 250–253.PubMedCrossRefGoogle Scholar
  12. 12.
    Root, D. and Wang, K. (1993) Silver-enhanced copper staining of protein blots. Analyt. Biochem. 209, 15–19.PubMedCrossRefGoogle Scholar
  13. 13.
    Patton, W., 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 immunoblotting. Analyt. Biochem. 220, 324–335.PubMedCrossRefGoogle Scholar
  14. 14.
    Shojaee, N., Patton, W., Lim, M., and Shepro, D. (1996) Pyrogallol red-molybdate; a reversible, metal chelate stain for detection of proteins immobilized on membrane supports. Electrophoresis 17, 687–695.PubMedCrossRefGoogle Scholar
  15. 15.
    Lim, M., Patton, W., Shojaee, N., and Shepro, D. (1996) A solid-phase metal chelate assay for quantifying total protein; resistance to chemical interference. Biotechniques 21, 888–897.PubMedGoogle Scholar
  16. 16.
    Lim, M., Patton, W., Shojaee, N., and Shepro, D. (1997) Comparison of a sensitive, solid-phase metal chelate protein assay with the bicinchoninic acid (BCA) assay. Am. Biotech. Lab. 15, 16–18.Google Scholar
  17. 17.
    Lim, M., Patton, W., Shojaee, N., Lopez, M., Spofford, K., and Shepro, D. (1997) A luminescent europium complex for the sensitive detection of proteins and nucleic acids immobilized on membrane supports. Analyt. Biochem. 245, 184–195.PubMedCrossRefGoogle Scholar
  18. 18.
    Chung, M. (1985) A specific iron stain for iron-binding proteins in polyacrylamide gels: application to transferrin and lactoferrin. Analyt. Biochem. 148, 498–502.PubMedCrossRefGoogle Scholar
  19. 19.
    Bickar, D. and Reid, P. (1992) A high-affinity protein stain for Western blots, tissue prints, and electrophoresis gels. Analyt. Biochem. 203, 109–115.PubMedCrossRefGoogle Scholar
  20. 20.
    Graham, G., Nairn, R., and Bates, G. (1978) Polyacrylamide gel staining with iron (II)-bathophenanthroline sulfonate. Analyt. Biochem. 88, 434–441.PubMedCrossRefGoogle Scholar
  21. 21.
    Zapolski, E., Gersten, D., and Ledley, R. (1982) [59Fe] ferrous bathophenathroline sulfonate: a radioactive stain for labeling proteins in situ in polyacrylamide gels. Analyt. Biochem. 123, 325–328.PubMedCrossRefGoogle Scholar
  22. 22.
    Berggren, K., Steinberg, T., Lauber, W., Carroll, J., Lopez, M., 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
  23. 23.
    Steinberg, T., Lauber, W., Berggren, K. Kemper, C., Yue, S., and Patton, W. (2000) Fluorescence detection of proteins in SDS-polyacrylamide Gels using environmentally benign, non-fixative, saline solution. Electrophoresis 21, 497–508.PubMedCrossRefGoogle Scholar
  24. 24.
    Steinberg, T., Chernokalskaya, E., Berggren, K., 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
  25. 25.
    Patton, W. (2000) A thousand points of light; the application of fluorescence detection technologies to two-dimensional gel electrophoresis and proteomics. Electrophoresis 21, 1123–1144.PubMedCrossRefGoogle Scholar
  26. 26.
    Patton, W. (2000) Making blind robots see; the synergy between fluorescent dyes and imaging devices in automated proteomics. BioTechniques 28, 944–957.PubMedGoogle Scholar
  27. 27.
    Berggren, K., Chernokalskaya, E., Steinberg, T., Kemper, C., Lopez, M., 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
  28. 28.
    Gosling, J. (1990) A decade of development in immunoassay methodology. Clin. Chem. 36, 1408–1427.PubMedGoogle Scholar
  29. 29.
    Harlow, E. and Lane, D. (1988) Antibodies; A Laboratory Manual. Cold Spring Harborm Laboratory Press, Cold Spring Harbor, New York, pp. 505.Google Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Wayne F. Patton
    • 1
  1. 1.Molecular Probes Inc.Eugene

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