Phosphate-binding Tag, a New Tool to Visualize Phosphorylated Proteins*S
By E. Kinoshita, Emiko Kinoshita-Kikuta, Keiko Takiyama, T. Koike. Molecular & Cellular Proteomics, 2006
1,076 citations recorded by the source corpus.
We introduce two methods for the visualization of phosphorylated proteins using alkoxide-bridged dinuclear metal (i.e. Zn2+ or Mn2+) complexes as novel phosphate-binding tag (Phos-tag) molecules. Both Zn2+- and Mn2+-Phos-tag molecules preferentially capture phosphomonoester dianions bound to Ser, Thr, and Tyr residues. One method is based on an ECL system using biotin-pendant Zn2+-Phos-tag and horseradish peroxidase-conjugated streptavidin. We demonstrate the electroblotting analyses of protein phosphorylation status by the phosphate-selective ECL signals. Another method is based on the mobility shift of phosphorylated proteins in SDS-PAGE with polyacrylamide-bound Mn2+-Phos-tag. Phosphorylated proteins in the gel are visualized as slower migration bands compared with corresponding dephosphorylated proteins. We demonstrate the kinase and phosphatase assays by phosphate affinity electrophoresis (Mn2+-Phos-tag SDS-PAGE).
Subfields: Molecular Biology, Radiology, Nuclear Medicine and Imaging, Spectroscopy
View Paper: https://www.semanticscholar.org/paper/8c2b7399ce80f8a36bb27a890640364d70292b37
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Citations in the corpus
- Handbook of ion channels (2015, neutral): “Another phosphate-binding reagent is Phos-tag (12).”
- Small molecule inhibition of the autophagy kinase ULK1 and identification of ULK1 substrates (2015, background): “To further explore ULK1 phosphorylation of Atg101 in cells, we examined themigration pattern of Atg101 during SDS-PAGE in gels containing the Phos-Tag reagent, a phosphate binding dinuclear metal complex that specifically retards the mobility of phosphoproteins (Kinoshita et al., 2006).”
- Development of an optimized backbone of FRET biosensors for kinases and GTPases (2011, methodology): “Phos-tag was obtained from the Phos-tag Consortium (Hiroshima, Japan; www.phos-tag.com).”