title: Tips on improving the efficiency of electrotransfer of target proteins from Phos-tag SDS-PAGE gel.
abstract: The sensitivity of Western blotting analysis after Phos-tag SDS-PAGE is occasionally inferior to that after normal (Phos-tag-free) SDS-PAGE under similar experimental conditions, possibly as a result of inefficient electrotransfer from the Phos-tag gel to the blotting membrane. We therefore present tips on improving the efficiency of electrotransfer of proteins in semidry and wet-tank blotting. When model samples containing several standard phosphoproteins were subjected to semidry blotting, their electrotransfer efficiencies after Phos-tag SDS-PAGE were markedly inferior to those of their dephosphorylated counterparts in the same gel. This was ameliorated by immersing the electrophoresed Phos-tag gel in a transfer buffer containing 1 mM EDTA for 30 min before electroblotting. Similarly, phosphoproteomes in crude cell extracts were inefficiently transferred by semidry blotting, but the efficiencies of their electrotransfer were improved by pretreatment with EDTA. In contrast, the efficiencies of wet-tank blotting of the same samples were not dependent on the degree of phosphorylation, and the efficiencies of electrotransfer of all proteins from Phos-tag gels were similar to those from normal gels. In some cases involving the use of a Phos-tag gel, addition of 0.1% w/v of SDS to the transfer buffer significantly improved the electrotransfer.
authors.affiliation: Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
authors.forename: Emiko
authors.initials: E
authors.lastname: Kinoshita-Kikuta
authors.forename: Eiji
authors.initials: E
authors.lastname: Kinoshita
authors.forename: Ayumi
authors.initials: A
authors.lastname: Matsuda
authors.forename: Tohru
authors.initials: T
authors.lastname: Koike
mesh_terms: D015153:Blotting, Western / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D010750:Phosphoproteins / Q000032:analysis* / Q000302:isolation & purification; D010766:Phosphorylation; D011725:Pyridines / Q000737:chemistry*
chemical_list: C514153:1,3-bis(bis(pyridin-2-ylmethyl)amino)propan-2-ol; D010750:Phosphoproteins; D011725:Pyridines
keywords: Phos-tag SDS-PAGE; Phosphoprotein; Semidry blotting; Technology; Western blotting; Wet-tank blotting