Detection of Proteins in Polyacrylamide Gels via Prelabeling by Isatoic Anhydride

  • Kazem Asadollahi
  • Saharnaz Rafiee
  • Gholamhossein RiaziEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1853)


Fluorescent methods for staining of proteins in polyacrylamide gels are obtaining increasing attention due to their advantages including high sensitivity and rapidness. In the present chapter we describe, step by step, a rapid and inexpensive fluorescent prelabeling method based on derivatization of proteins with isatoic anhydride. This method allows researchers to detect less than 2 ng of standard proteins per band in less than 15 min. Although this method is sensitive, inexpensive, rapid, compatible with most of common biological reagents and is able to detect proteins in crude cell extract, covalent binding of isatoic anhydride to protein lysine residues makes it unsuitable for processes in which post-electrophoresis analysis is required. Moreover, isatoic anhydride derivatization induces a small band broadening.

Key words

Isatoic anhydride Protein labeling Electrophoresis Staining 


  1. 1.
    Candiano G, Bruschi M, Musante L et al (2004) Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25:1327–1333. Scholar
  2. 2.
    Lawrence A, Besir H (2009) Staining of proteins in gels with Coomassie G-250 without organic solvent and acetic acid. J Vis Exp 30:2–4. Scholar
  3. 3.
    Tsolakos N, Techanukul T, Wallington A et al (2009) Comparison of two combinations of cyanine dyes for prelabelling and gel electrophoresis. Proteomics 9:1727–1730. Scholar
  4. 4.
    Chevalier F (2010) Standard dyes for total protein staining in gel-based proteomic analysis. Materials (Basel) 3:4784–4792. Scholar
  5. 5.
    Hrebicek T, Dürrschmid K, Auer N et al (2007) Effect of CyDye minimum labeling in differential gel electrophoresis on the reliability of protein identification. Electrophoresis 28:1161–1169. Scholar
  6. 6.
    Chevallet M, Luche S, Rabilloud T (2006) Silver staining of proteins in polyacrylamide gels. Nat Protoc 1:1852–1858. Scholar
  7. 7.
    Neuhoff V, Arold N, Taube D, Ehrhardt 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. Scholar
  8. 8.
    Chevalier F, Rofidal V, Vanova P et al (2004) Proteomic capacity of recent fluorescent dyes for protein staining. Phytochemistry 65:1499–1506. Scholar
  9. 9.
    Churchich JE (1993) Fluorescence properties of o-aminobenzoyl-labeled proteins. Anal Biochem 213:229–233CrossRefPubMedGoogle Scholar
  10. 10.
    Staiger RP, Miller EB (1953) Isatoic anhydride. IV. Reactions with various nucleophiles. J Org Chem 24:1214–1219. Scholar
  11. 11.
    Asadollahi K, Rafiee S, Riazi G (2016) Sensitive detection of proteins in polyacrylamide gel via isatoic anhydride derivatization: introduction of a low-cost fluorescent prelabeling procedure. Electrophoresis.

Copyright information

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

Authors and Affiliations

  • Kazem Asadollahi
    • 1
  • Saharnaz Rafiee
    • 1
  • Gholamhossein Riazi
    • 1
    Email author
  1. 1.Institute of Biochemistry and BiophysicsUniversity of TehranTehranIran

Personalised recommendations