title: Neutral Phosphate-Affinity SDS-PAGE system for profiling of protein phosphorylation.
abstract: In this chapter, we describe a standard protocol for phosphate-affinity SDS-PAGE that uses a dizinc(II) complex of the phosphate-binding molecule Phos-tag in conjunction with a neutral-pH gel system (Zn(2+-)Phos-tag SDS-PAGE) to detect shifts in the mobilities of phosphoproteins. A previous protocol for affinity electrophoresis that uses polyacrylamide-bound Mn(2+)-Phos-tag and Laemmli's buffer system under conditions of alkaline pH has limitations in separating certain phosphoproteins. The current protocol provides major improvements in separation and detection of various phosphorylated protein species. We here introduce two neutral-pH gel systems buffered with Bis-Tris-HCl and Tris-AcOH, respectively, for Zn(2+)-Phos-tag SDS-PAGE, and we also discuss their characteristics on the basis of comparative studies on phosphorylation profiling of proteins with a wide range of molecular masses. Each analytical procedure, from the beginning of gel preparation to the end of electrophoresis, requires 2.5-5 h with either buffer system.
authors.affiliation: Department of Functional Molecular Science, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, 734-8553, Japan.
authors.forename: Emiko
authors.initials: E
authors.lastname: Kinoshita-Kikuta
authors.forename: Eiji
authors.initials: E
authors.lastname: Kinoshita
authors.forename: Tohru
authors.initials: T
authors.lastname: Koike
mesh_terms: D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D020543:Proteome; D040901:Proteomics / Q000379:methods
chemical_list: D010750:Phosphoproteins; D011506:Proteins; D020543:Proteome