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Rapid and Sensitive Staining of Unfixed Proteins in Polyacrylamide Gels with Nile Red

  • Joan-Ramon Daban
  • Salvador Bartolomé
  • Antonio Bermúdez
  • F. Javier Alba
Protocol
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Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is one of the most powerful methods for protein analysis (1,2). However, the typical procedures for the detection of protein bands after SDS-PAGE, using the visible dye Coomassie Blue and silver staining, have several time-consuming steps and require the fixation of proteins in the gel. This chapter describes a rapid and very simple method for protein staining in SDS gels developed in our laboratory (3-5). The method is based on the fluorescent properties of the hydrophobic dye Nile red (9-diethylamino-5Hbenzo [α]phenoxazine-5-one; see Fig. 1), and allows the detection of<10 ng of unfixed protein per band about 5 min after the electrophoretic separation. Furthermore, it has been shown elsewhere (6,7) that, in contrast to the current staining methods, Nile red staining does not preclude the direct electroblotting of protein bands and does not interfere with further staining, immunodetection and sequencing (see  Chapter 50).

Keywords

Critical Micelle Concentration Sodium Sulfite Photographic Camera Photographic Negative Polaroid Film 
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.
    Andrews, A. T. (1986) Electrophoresis. Theory, Techniques, and Biochemical and Cinical Applications. Oxford University Press, Oxford, UK.Google Scholar
  2. 2.
    Allen, R. C. and Budowle, B. (1999) Protein Staining and Identification Techniques. Eaton Publishing, Natick, MA.Google Scholar
  3. 3.
    Daban, J.-R., Samsó, M., and Bartolomé, S. (1991) Use of Nile red as a fluorescent probe for the study of the hydrophobic properties of protein-sodium dodecyl sulfate complexes in solution. Analyt. Biochem. 199, 162–168.PubMedCrossRefGoogle Scholar
  4. 4.
    Daban, J.-R., Bartolomé, S., and Samsó, M. (1991) Use of the hydrophobic probe Nile red for the fluorescent staining of protein bands in sodium dodecyl sulfate-polyacrylamide gels. Analyt. Biochem. 199, 169–174.PubMedCrossRefGoogle Scholar
  5. 5.
    Alba, F. J., Bermúdez, A., Bartolomé, S., and Daban, J.-R. (1996) Detection of five nano-grams of protein by two-minute Nile red staining of unfixed SDS gels. BioTechniques. 21, 625–626.PubMedGoogle Scholar
  6. 6.
    Bermúdez, A., Daban, J.-R., Garcia, J. R., and Mendez, E. (1994) Direct blotting, sequencing and immunodetection of proteins after five-minute staining of SDS and SDS-treated IEF gels with Nile red. BioTechniques. 16, 621–624.PubMedGoogle Scholar
  7. 7.
    Alba, F. J. and Daban, J.-R. (1998) Rapid fluorescent monitoring of total protein patterns on sodium dodecyl sulfate-polyacrylamide gels and Western blots before immunodetection and sequencing. Electrophoresis. 19, 2407–2411.PubMedCrossRefGoogle Scholar
  8. 8.
    Greenspan, P. and Fowler, S. D. (1985) Spectrofluorometric studies of the lipid probe Nile red. J. Lipid Res. 26, 781–789.PubMedGoogle Scholar
  9. 9.
    Sackett, D. L. and Wolff, J. (1987) Nile red as polarity-sensitive fluorescent probe of hydrophobic protein surfaces. Analyt. Biochem. 167, 228–234.PubMedCrossRefGoogle Scholar
  10. 10.
    Sansó, M., Daban, J.-R., Hansen, S., and Jones, G. R. (1995) Evidence for sodium dodecyl sulfate/protein complexes adopting a necklace structure. Eur. J. Biochem. 232, 818–824.CrossRefGoogle Scholar
  11. 11.
    Alba, F. J., Bermúdez, A., and Daban, J.-R. (2001) Green-light transilluminator for the detection without photodamage of proteins and DNA labeled with different fluorescent dyes. Electrophoresis 22, 399–403.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Joan-Ramon Daban
    • 1
  • Salvador Bartolomé
    • 1
  • Antonio Bermúdez
    • 1
  • F. Javier Alba
    • 1
  1. 1.Departament de Bioquímica i Biologia MolecularUniversität Autónoma de BarcelonaBellaterra (Barcelona)Spain

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