Zn(II)-phos-tag SDS-PAGE for separation and detection of a DNA damage-related signaling large phosphoprotein

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.