Fluorescent Protein Visualization Immediately After Gel Electrophoresis Using an In-Gel Trichloroethanol Photoreaction with Tryptophan

  • Carol L. Ladner-Keay
  • Raymond J. Turner
  • Robert A. EdwardsEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1853)


SDS–polyacrylamide gel electrophoresis (SDS-PAGE) is one of the essential techniques in molecular biology and biochemistry laboratories and requires rapid visualization methods for efficient sample analysis. Proteins on polyacrylamide gels can be visualized within 5 min via the photoreaction of tryptophan with trichloroethanol. This process does not require protein fixation, staining, or destaining. In this method polyacrylamide gels are prepared by adding trichloroethanol before polymerization. After electrophoresis, the gel is immediately activated on a standard UV transilluminator and the fluorescently labeled proteins are imaged. The reaction is based on the photoreaction of trichloroethanol with tryptophan residues within the protein. This generates a visible blue-green fluorescence (∼500 nm) that is accurately imaged. Here we describe the preparation of Tris–glycine and Tris–tricine SDS–polyacrylamide gels with trichloroethanol and the photoreaction and visualization of tryptophan containing proteins.

Key words

Protein electrophoresis Fluorescent visualization Trichloroethanol Tryptophan photochemistry 



We thank Natural Sciences and Engineering Research Council of Canada (NSERC) for funding.


  1. 1.
    Westermeier R (2016) Electrophoresis in practice: a guide to methods and applications of DNA and protein separations. Wiley, New JerseyCrossRefGoogle Scholar
  2. 2.
    Gauci VJ, Wright EP, Coorssen JR (2011) Quantitative proteomics: assessing the spectrum of in-gel protein detection methods. J Chem Biol 4:3–29CrossRefPubMedGoogle Scholar
  3. 3.
    Dzandu JK, Johnson JF, Wise GE (1988) Sodium dodecyl sulfate-gel electrophoresis: staining of polypeptides using heavy metal salts. Anal Biochem 174:157–167CrossRefPubMedGoogle Scholar
  4. 4.
    Ladner CL, Yang J, Turner RJ, Edwards RA (2004) Visible fluorescent detection of proteins in polyacrylamide gels without staining. Anal Biochem 326:13–20CrossRefPubMedGoogle Scholar
  5. 5.
    Ladner CL, Khai T, Le M, Turner RJ, Edwards RA (2014) Excited state photoreaction between the indole side chain of tryptophan and halocompounds generates new fluorophores and unique modifications. Photochem Photobiol 90:1027–1033PubMedGoogle Scholar
  6. 6.
    Edwards RA, Jickling G, Turner RJ (2002) The light-induced reactions of tryptophan with halocompounds. Photochem Photobiol 75:362–368CrossRefPubMedGoogle Scholar
  7. 7.
    Bay DC, Budiman RA, Nieh M-P, Turner RJ (2010) Multimeric forms of the small multidrug resistance protein EmrE in anionic detergent. Biochim Biophys Acta 1798:526–535CrossRefPubMedGoogle Scholar
  8. 8.
    Ladner CL, Edwards RA, Schriemer DC, Turner RJ (2006) Identification of trichloroethanol visualized proteins from two-dimensional polyacrylamide gels by mass spectrometry. Anal Chem 78:2388–2396CrossRefPubMedGoogle Scholar
  9. 9.
    Colella AD, Chegenii N, Tea MN, Gibbins IL, Williams KA, Chataway TK (2012) Comparison of Stain-Free gels with traditional immunoblot loading control methodology. Anal Biochem 430:108–110CrossRefPubMedGoogle Scholar
  10. 10.
    Holzmueller W, Kulozik U (2016) Protein quantification by means of a stain-free SDS-PAGE technology without the need for analytical standards: verification and validation of the method. J Food Compos Anal 48:128–134CrossRefGoogle Scholar
  11. 11.
    Guertler A, Kunz N, Gomolka M, Hornhardt S, Friedl AA, McDonald K, Kohn JE, Posch A (2013) Stain-Free technology as a normalization tool in Western blot analysis. Anal Biochem 433:105–111CrossRefGoogle Scholar
  12. 12.
    Schagger H (2006) Tricine-SDS-PAGE. Nat Protoc 1:16–22CrossRefPubMedGoogle Scholar
  13. 13.
    Schägger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379CrossRefPubMedGoogle Scholar
  14. 14.
    Haider SR, Reid HJ, Sharp BL (2012) In: Kurien BT, Scofield RH (eds) Protein electrophoresis: methods and protocols. Humana Press, Totowa, NJ, pp 81–91CrossRefGoogle Scholar
  15. 15.
    Susnea I, Bernevic B, Wicke M, Ma L, Liu S, Schellander K, Przybylski M (2013) In: Cai Z, Liu S (eds) Applications of Maldi-Tof spectroscopy, vol 331, pp 37–54CrossRefGoogle Scholar
  16. 16.
    Montigny C, Decottignies P, Le Marechal P, Capy P, Bublitz M, Olesen C, Moller JV, Nissen P, le Maire M (2014) S-Palmitoylation and S-oleoylation of rabbit and pig sarcolipin. J Biol Chem 289:33850–33861CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Laurie KJ, Dave A, Straga T, Souzeau E, Chataway T, Sykes MJ, Casey T, Teo T, Pater J, Craig JE et al (2013) Identification of a novel oligomerization disrupting mutation in CRYA associated with congenital cataract in a south Australian family. Hum Mutat 34:435–438CrossRefPubMedGoogle Scholar
  18. 18.
    Gilda JE, Gomes AV (2015) In: Posch A (ed) Proteomic profiling: methods and protocols, vol 1295, pp 381–391Google Scholar
  19. 19.
    Raykin J, Snider E, Bheri S, Mulvihill J, Ethier CR (2017) A modified gelatin zymography technique incorporating total protein normalization. Anal Biochem 521:8–10CrossRefPubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Carol L. Ladner-Keay
    • 1
  • Raymond J. Turner
    • 2
  • Robert A. Edwards
    • 2
    Email author
  1. 1.Department of Chemical and Materials EngineeringUniversity of AlbertaEdmontonCanada
  2. 2.Department of Biological SciencesUniversity of CalgaryCalgaryCanada

Personalised recommendations