Chapter title |
Zn(II)–Phos-Tag SDS-PAGE for Separation and Detection of a DNA Damage-Related Signaling Large Phosphoprotein
|
---|---|
Chapter number | 9 |
Book title |
ATM Kinase
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-6955-5_9 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6953-1, 978-1-4939-6955-5
|
Authors |
Eiji Kinoshita, Emiko Kinoshita-Kikuta, Tohru Koike |
Editors |
Sergei V. Kozlov |
Abstract |
In this chapter, we provide a standard protocol for phosphate-affinity sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Zn(2+)-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 Zn(2+)-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. |
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