Abstract
We introduce two methods for the visualization of phosphorylated proteins using alkoxide-bridged dinuclear metal (i.e. Zn(2+) or Mn(2+)) complexes as novel phosphate-binding tag (Phos-tag) molecules. Both Zn(2+)- and Mn(2+)-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 Zn(2+)-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 Mn(2+)-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 (Mn(2+)-Phos-tag SDS-PAGE).
Highlights
We introduce two methods for the visualization of phosphorylated proteins using alkoxide-bridged dinuclear metal (i.e. Zn2؉ or Mn2؉) complexes as novel phosphatebinding tag (Phos-tag) molecules
As the first example, phosphorylated proteins (i.e. ␣-casein, -casein, ovalbumin, and pepsin) spotted on a PVDF membrane were detected at nanogram levels using an ECL system and a 4:1 complex of biotin-pendant Zn2ϩ-Phos-tag and HRP-SA without a blocking treatment of the membrane
In the absence of the zinc(II) ions, no ECL signals were detected on the spots of phosphorylated proteins
Summary
Phos-tag, phosphate-binding tag; AP, alkaline phosphatase; EGF, epidermal growth factor; HRP, horseradish peroxidase; HRP-SA, horseradish peroxidase-conjugated streptavidin; TC-PTP, T-cell protein tyrosine phosphatase; SPR, surface plasmon resonance; CBB, Coomassie Brilliant Blue R-250; TE-. The anion selectivity indexes of the phenyl phosphate dianion against SO42Ϫ, CH3COOϪ, ClϪ, and the bisphenyl phosphate monoanion at 25 °C are 5.2 ϫ 103, 1.6 ϫ 104, 8.0 ϫ 105, and Ͼ2 ϫ 106, respectively These findings have contributed to the development of procedures for MALDI-TOF-MS for the analysis of phosphorylated compounds (e.g. phosphopeptides and phospholipids) [5,6,7], IMAC for the separation of phosphopeptides and phosphorylated proteins [8], and surface plasmon resonance (SPR) analysis for reversible peptide phosphorylation [9]. One is the chemiluminescence detection of whole phosphorylated proteins on electroblotting membranes using biotinylated Zn2ϩ-Phos-tag and HRP-SA Another is simple SDS-PAGE for the separation of a phosphorylated protein and the corresponding nonphosphorylated one where polyacrylamide-bound Mn2ϩ-Phos-tag was used as a phosphate-binding moiety
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