Abstract
Western blotting is a commonly used laboratory technique for semi-quantifying protein amounts. It is important when quantifying protein expression to account for differences in the amount of total protein loaded onto the gel using a loading control. Common loading controls include housekeeping proteins, such as β-actin or GAPDH, quantified by Western blot, or total protein, quantified using a stain such as Coomassie Brilliant Blue or Ponceau S. A more recently developed method for total protein quantification utilizes stain-free technology, which has a linear dynamic detection range and allows for protein detection on both gels and membranes. Here, we describe the theory and use of stain-free gels for total protein quantification and normalization of Western blots.
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References
Kurien BT, Scofield RH (2006) Western blotting. Methods 38:283–293
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354
Murphy RM, Lamb GD (2013) Important considerations for protein analyses using antibody based techniques: down-sizing Western blotting up-sizes outcomes. J Physiol 591:5823–5831
Posch A, Kohn J, Oh K, Hammond M, Liu N (2013) V3 stain-free workflow for a practical, convenient, and reliable total protein loading control in western blotting. J Vis Exp 82:50948
Gilda JE, Gomes AV (2013) Stain-Free total protein staining is a superior loading control to beta-actin for Western blots. Anal Biochem 440:186–188
Dittmer A, Dittmer J (2006) Beta-actin is not a reliable loading control in Western blot analysis. Electrophoresis 27:2844–2845
Eaton SL, Roche SL, Llavero Hurtado M, Oldknow KJ, Farquharson C, Gillingwater TH, Wishart TM (2013) Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting. PLoS One 8:e72457
Ruan W, Lai M (2007) Actin, a reliable marker of internal control? Clin Chim Acta 385:1–5
Schmittgen TD, Zakrajsek BA (2000) Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J Biochem Biophys Methods 46:69–81
Rodriguez-Mulero S, Montanya E (2005) Selection of a suitable internal control gene for expression studies in pancreatic islet grafts. Transplantation 80:650–652
Zeng L, Guo J, Xu HB, Huang R, Shao W, Yang L, Wang M, Chen J, Xie P (2013) Direct Blue 71 staining as a destaining-free alternative loading control method for Western blotting. Electrophoresis 34:2234–2239
Aldridge GM, Podrebarac DM, Greenough WT, Weiler IJ (2008) The use of total protein stains as loading controls: an alternative to high-abundance single-protein controls in semi-quantitative immunoblotting. J Neurosci Methods 172:250–254
Gurtler 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
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
Acknowledgement
This work was supported by the National Institutes of Health Grants HL080101 and HL096819.
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Gilda, J.E., Gomes, A.V. (2015). Western Blotting Using In-Gel Protein Labeling as a Normalization Control: Stain-Free Technology. In: Posch, A. (eds) Proteomic Profiling. Methods in Molecular Biology, vol 1295. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2550-6_27
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DOI: https://doi.org/10.1007/978-1-4939-2550-6_27
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2549-0
Online ISBN: 978-1-4939-2550-6
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