Journal
PROTEOMICS
Volume 11, Issue 2, Pages 319-323Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/pmic.201000472
Keywords
Bis-tris; Phos-tag; Phosphoprotein; Phosphorylation; SDS-PAGE; Technology
Funding
- Japan Society of the Promotion of Science [22590037]
- Ministry of Education, Culture, Sports, Science, and Technology [22790037]
- Takeda Science Foundation
- Ube Foundation
- Grants-in-Aid for Scientific Research [22590037] Funding Source: KAKEN
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We describe an improved Phos-tag SDS-PAGE (Zn(2+)-Phos-tag SDS-PAGE) using a dizinc(II) complex of Phos-tag acrylamide in conjunction with a Bis-tris-buffered neutral-pH gel system to detect shifts in the mobility of phosphoproteins. An existing technique (Mn(2+)-Phos-tag SDS-PAGE) using a polyacrylamide-bound Mn(2+)-Phos-tag and a conventional Laemmli's buffer system under alkaline pH conditions has limitations for separating certain phosphoproteins. The major improvements were demonstrated by visualizing novel up-shifted bands of commercially available pepsin, recombinant Tau treated in vitro with tyrosine kinases, and endogeneous beta-catenin in whole-cell lysates. Additionally, the Zn(2+)-Phos-tag SDS-PAGE gels showed better long-term stability than the Mn(2+)-Phos-tag SDS-PAGE gels. We can therefore provide a simple, convenient, and more reliable homemade gel system for phosphate-affinity SDS-PAGE.
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