Revisit of Imidazole-Zinc Reverse Stain for Protein Polyacrylamide Gel Electrophoresis

  • Han-Min ChenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1853)


Imidazole-zinc reverse stain (ZN stain) is known for high sensitivity, ease of use, and cost-effective feature. ZN stain is compatible to many experiments of which those are proteomics-related in particular. Here, we describe the ZN staining procedures and the subsequent procedures incorporated in detail, along with the improvements of setup in aspects of visualization and documentation for postprocessing ZN stained gel images.

Key words

Imidazole-zinc reverse stain SDS-PAGE Native-PAGE Electroblotting Electroelution Mass spectrometry 



This work was supported by the grants from Catholic Fu-Jen University (9991A02-109931041001-2) and National Science Council of the Republic of China (NSC 99-2627-E-030-001). We thank Dr. Kuo Cheng-Yi for linguistic edition, and Mr. Tsai Tzung-Lin and Dr. Wu Szu-Yu for providing gel images.


  1. 1.
    Fernandez-Patron C, Castellanos-Serra L (1990) Abstract Booklet In: Eight international conference on methods on protein sequence analysis, Kiruna, Sweden, July 1–6, 1990Google Scholar
  2. 2.
    Fernandez-Patron C, Castellanos-Serra L, Rodriguez P (1992) Reverse staining of sodium dodecyl sulfate polyacrylamide gels by imidazole-zinc salts: sensitive detection of unmodified proteins. BioTechniques 12:564573PubMedGoogle Scholar
  3. 3.
    Ferreras M, Gavilanes JG, Garcia-Segura JM (1993) A permanent Zn2+ reverse staining method for the detection and quantification of proteins in polyacrylamide gels. Anal Biochem 213:206212CrossRefPubMedGoogle Scholar
  4. 4.
    Hardy E, Pupo E, Casalvilla R et al (1996) Negative staining with zinc-imidazole of gel electrophoresis-separated nucleic acids. Electrophoresis 17:15371541CrossRefPubMedGoogle Scholar
  5. 5.
    Hardy E, Sosa AE, Pupo E et al (1996) Zinc-imidazole positive: a new method for DNA detection after electrophoresis on agarose gels not interfering with DNA biological integrity. Electrophoresis 17:2629CrossRefPubMedGoogle Scholar
  6. 6.
    Hardy E, Pupo E, Castellanos-Serra L et al (1997) Sensitive reverse staining of bacterial lipopolysaccharides on polyacrylamide gels by using zinc and imidazole salts. Anal Biochem 244:2832CrossRefPubMedGoogle Scholar
  7. 7.
    Pupo E, Lopez CM, Alonso M et al (2000) High-efficiency passive elution of bacterial lipopolysaccharides from polyacrylamide gels. Electrophoresis 21:526530CrossRefPubMedGoogle Scholar
  8. 8.
    Castellanos-Serra L, Hardy E (2001) Detection of biomolecules in electrophoresis gels with salts of imidazole and zinc II: a decade of research. Electrophoresis 22:864873CrossRefPubMedGoogle Scholar
  9. 9.
    Hardy E, Castellanos-Serra LR (2004) “Reverse-staining” of biomolecules in electrophoresis gels: analytical and micropreparative applications. Anal Biochem 328:113CrossRefPubMedGoogle Scholar
  10. 10.
    Lin CY, Wang V, Shui HA et al (2009) A comprehensive evaluation of imidazole-zinc reverse stain for current proteomic researches. Proteomics 9:696709CrossRefPubMedGoogle Scholar
  11. 11.
    Scheler C, Lamer S, Pan Z et al (1998) Peptide mass fingerprint sequence coverage from differently stained proteins on two-dimensional electrophoresis patterns by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Electrophoresis 19:918927CrossRefPubMedGoogle Scholar
  12. 12.
    Castellanos-Serra L, Proenza W, Huerta V et al (1999) Proteome analysis of polyacrylamide gel-separated proteins visualized by reversible negative staining using imidazole-zinc salts. Electrophoresis 20:732737CrossRefPubMedGoogle Scholar
  13. 13.
    Gulin S, Pupo E, Schweda EK et al (2003) Linking mass spectrometry and slab-polyacrylamide gel electrophoresis by passive elution of lipopolysaccharides from reverse-stained gels: analysis of gel-purified lipopolysaccharides from Haemophilus influenzae strain Rd. Anal Chem 75:49184924CrossRefPubMedGoogle Scholar
  14. 14.
    Fernandez-Patron C, Calero M, Collazo PR et al (1995) Protein reverse staining: high-efficiency microanalysis of unmodified proteins detected on electrophoresis gels. Anal Biochem 224:203211CrossRefPubMedGoogle Scholar
  15. 15.
    Fernandez-Patron C, Castellanos-Serra L, Hardy E et al (1998) Understanding the mechanism of the zinc-ion stains of biomacromolecules in electrophoresis gels: generalization of the reverse-staining technique. Electrophoresis 19:23982406CrossRefPubMedGoogle Scholar
  16. 16.
    Lin CY, Huang HM, Chen HM (2006) Use of backlit light plate to enhance visualization of imidazole-zinc reverse stained gels. BioTechniques 41:560 562, 564CrossRefPubMedGoogle Scholar
  17. 17.
    Wu HC, Yen CC, Tsui WH et al (2010) A red line not to cross: evaluating the limitation and properness of gel image tuning procedures. Anal Biochem 396:4250CrossRefPubMedGoogle Scholar
  18. 18.
    Westermeir R, Naven T (2002) Proteomics in practice. Wiley-VCH Verlag-GmbH, WeinheimCrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Life ScienceCatholic Fu-Jen UniversityNew Taipei CityTaiwan

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