The Roles of Acetic Acid and Methanol During Fixing and Staining Proteins in an SDS–Polyacrylamide Electrophoresis Gel

  • J. P. Dean GoldringEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1853)


After SDS–polyacrylamide gel electrophoresis proteins are “fixed” in the gel to prevent dispersion of the proteins and visualized by staining with a chromogenic dye. Dyes like Coomassie Blue R-250, Amido Black, and Direct Red 81 are usually dissolved in an acetic acid–methanol–water mixture. During staining the dye solvent mixture infuses the gel and interacts with the protein. Acetic acid and methanol denature the protein and provide an acidic environment enhancing the interactions with dyes. After staining, the dye that is in the gel and not bound to the protein, is removed using the solvent medium the dyes were dissolved in. Over 2–3 h the solution surrounding the gel becomes colored, the gel becomes lighter and the protein bands remain dark and the contrast against the surrounding gel improves. This chapter describes how each of the individual components in the dye solution interact with the protein resulting in a stained protein band in a clear SDS–polyacrylamide electrophoresis gel.

Key words

Acetic acid Methanol Fixing Staining Coomassie Blue SDS-PAGE 


  1. 1.
    Wirth PJ, Romano A (1995) Staining methods in gel-electrophoresis, including the use of multiple detection methods. J Chromatogr A 698:123–143CrossRefPubMedGoogle Scholar
  2. 2.
    Merril CR (1990) Gel-staining techniques. Methods Enzymol 182:477–488CrossRefPubMedGoogle Scholar
  3. 3.
    Schaffner W, Weissman C (1973) Rapid, sensitive, and specific method for determination of protein in dilute-solution. Anal Biochem 56:502–514CrossRefPubMedGoogle Scholar
  4. 4.
    Achilonu I, Goldring JPD (2010) Direct red 81 and amido black stain proteins in polyacrylamide electrophoresis gels within 10 min. Anal Biochem 400:139–141CrossRefPubMedGoogle Scholar
  5. 5.
    Alleger WW (1894) Formalin. Proceedings of the American Microscopical Society, Vol 15(3), Sixteenth Annual Meeting, Part III (March 1894), pp 192–197Google Scholar
  6. 6.
    Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  7. 7.
    Giourgiou CD, Grintsalis K, Zervoudakis G, Papapostolou I (2008) Mechanism of Coomassie brilliant blue G-250 binding to proteins: a hydrophobic assay for nanogram quantities of proteins. Anal Bioanal Chem 391:391–403CrossRefGoogle Scholar
  8. 8.
    Scewczyk B, Kozlof KK (1985) A method for the efficient blotting of strongly basic proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose. Anal Biochem 150:403–407CrossRefGoogle Scholar
  9. 9.
    Ranganathan V, De PK (1996) Western blot of proteins from Coomassie Blue stained gels. Anal Biochem 234:102–104CrossRefPubMedGoogle Scholar
  10. 10.
    Phang T, Ji I, Ji TH (1996) No need of acetic acid for processing polyacrylamide gels. Anal Biochem 234:96–97CrossRefPubMedGoogle Scholar
  11. 11.
    Kaplan B, Pras M (1990) Removal of sodium dodecyl sulfate from proteins isolated by sodium sulfate polyacrylamide gel electrophoresis. Biomed Chromatogr 4(2):89–90CrossRefPubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.BiochemistryUniversity of KwaZulu-NatalScottsvilleSouth Africa

Personalised recommendations