Abstract
In this chapter, we provide a standard protocol for phosphate-affinity sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Zn2+–Phos-tag SDS-PAGE). This technique uses a dizinc(II) complex of the phosphate-binding molecule Phos-tag in conjunction with a neutral-pH gel system, Tris [tris(hydroxymethyl)aminomethane], and acetic acid (Tris–AcOH), to detect shifts in the mobility of phosphorylated ataxia telangiectasia-mutated (ATM) kinase. This protocol, which employs a 3% (w/v) polyacrylamide gel strengthened with 0.5% (w/v) agarose, permits the separation of larger phosphoproteins with molecular masses in the order of 200 kDa over a period of approximately 4 h. Subsequently, multiple phosphorylated forms of high-molecular-mass ATM kinase (350 kDa) can be clearly detected via immunoblotting as multiple upshifted migration bands on the Zn2+–Phos-tag SDS-PAGE gel. The procedure described in this protocol requires a completion time of approximately 5 h from the beginning of gel preparation to the end of electrophoresis.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Kinoshita E, Takahashi M, Takeda H et al (2004) Recognition of phosphate monoester dianion by an alkoxide-bridged dinuclear zinc(II) complex. Dalton Trans 1189–1193. http://pubs.rsc.org/en/Content/ArticleLanding/2004/DT/b400269e#!divAbstract
Kinoshita E, Kinoshita-Kikuta E, Takiyama K et al (2006) Phosphate-binding tag, a new tool to visualize phosphorylated proteins. Mol Cell Proteomics 5:749–757
Kinoshita-Kikuta E, Aoki Y, Kinoshita E et al (2007) Label-free kinase profiling using phosphate affinity polyacrylamide gel electrophoresis. Mol Cell Proteomics 6:356–366
Kinoshita E, Kinoshita-Kikuta E, Matsubara M et al (2008) Separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate-affinity SDS-PAGE. Proteomics 8:2994–3003
Kinoshita E, Kinoshita-Kikuta E, Matsubara M et al (2009) Two-dimensional phosphate-affinity gel electrophoresis for the analysis of phosphoprotein isotypes. Electrophoresis 30:550–559
Kinoshita E, Kinoshita-Kikuta E, Ujihara H et al (2009) Mobility shift detection of phosphorylation on large proteins using a Phos-tag SDS-PAGE gel strengthened with agarose. Proteomics 9:4098–4101
Kinoshita E, Kinoshita-Kikuta E, Koike T (2009) Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE. Nat Protoc 4:1513–1521
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Kinoshita E, Kinoshita-Kikuta E, Koike T (2009) Phosphate-affinity gel electrophoresis using a Phos-tag molecule for phosphoproteome study. Curr Proteomics 6:104–121
Yamada S, Nakamura H, Kinoshita E et al (2007) Separation of a phosphorylated histidine protein using phosphate affinity polyacrylamide gel electrophoresis. Anal Biochem 360:160–162
Sugiyama M, Sugiyama Y, Hatano N et al (2010) The DNA-binding activity of mouse DNA methyltransferase 1 is regulated by phosphorylation with casein kinase 1δ/ε. Biochem J 427:489–497
Ishiai M, Kitao H, Smogorzewska A et al (2008) FANCI phosphorylation functions as a molecular switch to turn on the Fanconi anemia pathway. Nat Struct Mol Biol 15:1138–1146
Kinoshita E, Kinoshita-Kikuta E (2011) Improved Phos-tag SDS-PAGE under neutral pH conditions for advanced protein phosphorylation profiling. Proteomics 11:319–323
Kinoshita E, Kinoshita-Kikuta E, Koike T (2012) Phos-tag SDS-PAGE systems for phosphorylation profiling of proteins with a wide range of molecular masses under neutral pH conditions. Proteomics 12:192–202
Kinoshita-Kikuta E, Kinoshita E, Koike T (2012) Separation and identification of four distinct serine-phosphorylation states of ovalbumin by Phos-tag affinity electrophoresis. Electrophoresis 33:849–855
Kinoshita E, Kinoshita-Kikuta E, Shiba A et al (2014) Profiling of protein thiophosphorylation by Phos-tag affinity electrophoresis: evaluation of adenosine 5′-O-(3-thiotriphosphate) as a phosphoryl donor in protein kinase reactions. Proteomics 14:668–679
Kinoshita-Kikuta E, Kinoshita E, Eguchi Y et al (2015) Functional characterization of the receiver domain for phosphorelay Ccntrol in hybrid sensor kinases. PLoS One 10:e0132598
Kinoshita-Kikuta E, Kinoshita E, Eguchi Y et al (2016) Validation of cis and trans modes in multistep phosphotransfer signaling of bacterial tripartite sensor kinases by using Phos-tag SDS-PAGE. PLoS One 11:e0148294
Sepulveda P, Binner JGP (2001) Persulfate–amine initiation systems for gelcasting of ceramic foams. Chem Mater 13:4065–4070
Kinoshita-Kikuta E, Kinoshita E, Matsuda A et al (2014) Tips on improving the efficiency of electrotransfer of target proteins from Phos-tag SDS-PAGE gel. Proteomics 14:2437–2442
Kozlov SV, Graham ME, Peng C et al (2006) Involvement of novel autophosphorylation sites in ATM activation. EMBO J 25:3504–3514
Kozlov SV, Graham ME, Jakob B et al (2011) Autophosphorylation and ATM activation: additional sites add to the complexity. J Biol Chem 286:9107–9119
Kinoshita-Kikuta E, Kinoshita E, Koike T (2012) A laborsaving, timesaving, and more reliable strategy for separation of low-molecular-mass phosphoproteins in Phos-tag affinity electrophoresis. Int J Chem (Mumbai, India) 4(5):1–8
Acknowledgments
This work was supported in part by KAKENHI Grant no. 25293005 to EK, no. 25560417 to EK, no. 15K07887 to EK-K, and no. 26460036 to TK, and by research grants from the Takeda Science Foundation to EK and EK-K.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Kinoshita, E., Kinoshita-Kikuta, E., Koike, T. (2017). Zn(II)–Phos-Tag SDS-PAGE for Separation and Detection of a DNA Damage-Related Signaling Large Phosphoprotein. In: Kozlov, S. (eds) ATM Kinase. Methods in Molecular Biology, vol 1599. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6955-5_9
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6955-5_9
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6953-1
Online ISBN: 978-1-4939-6955-5
eBook Packages: Springer Protocols