Abstract
Here, we describe the detailed step-by-step protocol for detection of phosphoproteins in two-dimensional difference gel electrophoresis (DIGE) gels. A standard DIGE protocol is combined with subsequent post-staining with phosphospecific fluorescent dye. The combination of these two methods complements DIGE-based proteome profiling by fluorescence detection of phosphoproteins in the same gel providing additional possibility for sensitive and accurate quantification of the differentially regulated phosphoproteins in biological samples.
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References
Görg A, Weiss W, Dunn MJ (2004) Current two-dimensional electrophoresis technology for proteomics. Proteomics 4(12):3665–3685. doi:10.1002/pmic.200401031
Patton WF, Beechem JM (2002) Rainbow’s end: the quest for multiplexed fluorescence quantitative analysis in proteomics. Curr Opin Chem Biol 6(1):63–69
Ünlü M, Morgan ME, Minden JS (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18(11):2071–2077. doi:10.1002/elps.1150181133
Mackintosh JA, Choi HY, Bae SH, Veal DA, Bell PJ, Ferrari BC, Van Dyk DD, Verrills NM, Paik YK, Karuso P (2003) A fluorescent natural product for ultra sensitive detection of proteins in one-dimensional and two-dimensional gel electrophoresis. Proteomics 3(12):2273–2288. doi:10.1002/pmic.200300578
Berggren KN, Schulenberg B, Lopez MF, Steinberg TH, Bogdanova A, Smejkal G, Wang A, Patton WF (2002) An improved formulation of SYPRO Ruby protein gel stain: comparison with the original formulation and with a ruthenium II tris (bathophenanthroline disulfonate) formulation. Proteomics 2(5):486–498. doi:10.1002/1615-9861(200205)2:5<486::AID-PROT486>3.0.CO;2-X
Rabilloud T, Strub JM, Luche S, van Dorsselaer A, Lunardi J (2001) A comparison between Sypro Ruby and ruthenium II tris (bathophenanthroline disulfonate) as fluorescent stains for protein detection in gels. Proteomics 1(5):699–704. doi:10.1002/1615-9861(200104)1:5<699::AID-PROT699>3.0.CO;2-C
Steinberg TH, Agnew BJ, Gee KR, Leung WY, Goodman T, Schulenberg B, Hendrickson J, Beechem JM, Haugland RP, Patton WF (2003) Global quantitative phosphoprotein analysis using multiplexed proteomics technology. Proteomics 3(7):1128–1144. doi:10.1002/pmic.200300434
Stasyk T, Morandell S, Bakry R, Feuerstein I, Huck CW, Stecher G, Bonn GK, Huber LA (2005) Quantitative detection of phosphoproteins by combination of two-dimensional difference gel electrophoresis and phosphospecific fluorescent staining. Electrophoresis 26(14):2850–2854. doi:10.1002/elps.200500026
Stasyk T, Schiefermeier N, Skvortsov S, Zwierzina H, Peränen J, Bonn GK, Huber LA (2007) Identification of endosomal epidermal growth factor receptor signaling targets by functional organelle proteomics. Mol Cell Proteomics 6(5):908–922. doi:10.1074/mcp.M600463-MCP200
Orsatti L, Forte E, Tomei L, Caterino M, Pessi A, Talamo F (2009) 2-D difference in gel electrophoresis combined with pro-Q Diamond staining: a successful approach for the identification of kinase/phosphatase targets. Electrophoresis 30(14):2469–2476. doi:10.1002/elps.200800780
Mattei B, Spinelli F, Pontiggia D, De Lorenzo G (2016) Comprehensive analysis of the membrane phosphoproteome regulated by oligogalacturonides in Arabidopsis thaliana. Front Plant Sci 7:1107. doi:10.3389/fpls.2016.01107
Wessel D, Flügge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138(1):141–143
Lamanda A, Zahn A, Roder D, Langen H (2004) Improved ruthenium II tris (bathophenantroline disulfonate) staining and destaining protocol for a better signal-tobackground ratio and improved baseline resolution. Proteomics 4(3):599–608. doi:10.1002/pmic.200300587
de Araùjo ME, Huber LA, Stasyk T (2008) Isolation of endocitic organelles by density gradient centrifugation. Methods Mol Biol 424:317–331. doi:10.1007/978-1-60327-064-9_25
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Stasyk, T., Huber, L.A. (2018). DIGE-Based Phosphoproteomic Analysis. In: Ohlendieck, K. (eds) Difference Gel Electrophoresis. Methods in Molecular Biology, vol 1664. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7268-5_7
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DOI: https://doi.org/10.1007/978-1-4939-7268-5_7
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-7268-5
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