Abstract
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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Westermeier R (2016) Electrophoresis in practice: a guide to methods and applications of DNA and protein separations. Wiley, New Jersey
Gauci VJ, Wright EP, Coorssen JR (2011) Quantitative proteomics: assessing the spectrum of in-gel protein detection methods. J Chem Biol 4:3–29
Dzandu JK, Johnson JF, Wise GE (1988) Sodium dodecyl sulfate-gel electrophoresis: staining of polypeptides using heavy metal salts. Anal Biochem 174:157–167
Ladner CL, Yang J, Turner RJ, Edwards RA (2004) Visible fluorescent detection of proteins in polyacrylamide gels without staining. Anal Biochem 326:13–20
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 90:1027–1033
Edwards RA, Jickling G, Turner RJ (2002) The light-induced reactions of tryptophan with halocompounds. Photochem Photobiol 75:362–368
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 1798:526–535
Ladner CL, Edwards RA, Schriemer DC, Turner RJ (2006) Identification of trichloroethanol visualized proteins from two-dimensional polyacrylamide gels by mass spectrometry. Anal Chem 78:2388–2396
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 430:108–110
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 48:128–134
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 433:105–111
Schagger H (2006) Tricine-SDS-PAGE. Nat Protoc 1:16–22
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 166:368–379
Haider SR, Reid HJ, Sharp BL (2012) In: Kurien BT, Scofield RH (eds) Protein electrophoresis: methods and protocols. Humana Press, Totowa, NJ, pp 81–91
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 37–54
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 289:33850–33861
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 34:435–438
Gilda JE, Gomes AV (2015) In: Posch A (ed) Proteomic profiling: methods and protocols, vol 1295, pp 381–391
Raykin J, Snider E, Bheri S, Mulvihill J, Ethier CR (2017) A modified gelatin zymography technique incorporating total protein normalization. Anal Biochem 521:8–10
Acknowledgments
We thank Natural Sciences and Engineering Research Council of Canada (NSERC) for funding.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Ladner-Keay, C.L., Turner, R.J., Edwards, R.A. (2018). Fluorescent Protein Visualization Immediately After Gel Electrophoresis Using an In-Gel Trichloroethanol Photoreaction with Tryptophan. In: Kurien, B., Scofield, R. (eds) Protein Gel Detection and Imaging. Methods in Molecular Biology, vol 1853. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8745-0_22
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8745-0_22
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8744-3
Online ISBN: 978-1-4939-8745-0
eBook Packages: Springer Protocols