A Laborsaving, Timesaving, and More Reliable Strategy for Separation of Low-Molecular-Mass Phosphoproteins in Phos-tag Affinity Electrophoresis


  •  Emiko Kinoshita-Kikuta    
  •  Eiji Kinoshita    
  •  Tohru Koike    

Abstract

Reversible phosphorylation is a key signaling mechanism for modulating the functional properties of proteins involved in numerous cellular events. Abnormal protein phosphorylation causes many human diseases. Experimental procedures for the determination of the phosphorylation status of certain proteins are therefore very important in relation to studies on a diverse range of physiological and pathological processes. We have previously reported a novel phosphate-affinity sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technique using a dizinc(II) complex of the phosphate-binding ligand Phos-tag in conjunction with a neutral-pH gel system to detect shifts in the mobilities of phosphoproteins (Zn2+–Phos-tag SDS-PAGE). However, this handmade gel-based procedure is often laborious and time-consuming to perform, and requires a skillful analyst. More recently, SuperSep Phos-tag precast gel has been developed on the basis of the Zn2+–Phos-tag SDS-PAGE methodology. This novel ready-to-use system employs a neutral-pH gel containing 12.5% (w/v) polyacrylamide and the immobilized Zn2+–Phos-tag (50 µM), which is generally used in conjunction with a Tris–glycine-based electrophoretic running buffer. We examined the potential usage of a Tris–N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine (Tris–Tricine) buffer as an alternative running buffer for the SuperSep Phos-tag precast gel system in the analysis of low-molecular-mass phosphoproteins. Compared with Tris–glycine, the Tris–Tricine running buffer improved the resolution of 8.8-35 kDa phosphoproteins and phosphopeptides. We can therefore provide a laborsaving, timesaving, and more reliable strategy for separation of low-molecular-mass phosphoproteins in Phos-tag affinity electrophoresis.



This work is licensed under a Creative Commons Attribution 4.0 License.
  • ISSN(Print): 1916-9698
  • ISSN(Online): 1916-9701
  • Started: 2009
  • Frequency: semiannual

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