Improved Phos‐tag SDS‐PAGE under neutral pH conditions for advanced protein phosphorylation profiling

Abstract

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 β-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.

The General Stress Response σS Is Regulated by a Partner Switch in the Gram-negative Bacterium Shewanella oneidensis

“…CrsA and CrsR D3 (10 M each) were incubated either alone or together (20 l final volume) in 50 mM Tris-HCl, pH 7.6, 100 mM NaCl, 20 mM MgCl 2 , 1 mM DTT and in the presence or absence of 1 mM ATP, for 3 h at 30°C. Samples were subjected to Phos-Tag TM -10% PAGE prepared as previously described (58,59). Proteins were revealed by Coomassie Blue staining (Instant Blue-Expedeon).…”

Signal Transducer and Activator of Transcription 3 (STAT3) Protein Suppresses Adenoma-to-carcinoma Transition in Apc/+ Mice via Regulation of Snail-1 (SNAI) Protein Stability

“…Because STAT3 deletion or knockdown did not significantly affect the expression of GSK3␤ (Fig. 5C), we examined whether STAT3 regulates activation (or phosphorylation) of GSK3␤ by Phostag SDS-PAGE (13,32). Whereas there was no noticeable difference in GSK3␤ expression level between control and STAT3-deleted cells in a traditional SDS-PAGE analysis, the putative phosphorylated (retarded in mobility) GSK3␤ level was much higher in STAT3-deleted cells compared with that in control cells (Fig.…”

Inhibition of Acetyl Phosphate-dependent Transcription by an Acetylatable Lysine on RNA Polymerase

“…To monitor phospho-CpxR, we took advantage of a previously reported method used for the detection of phosphorylated proteins (27,28), including bacterial response regulators (11,29). This method uses a dinuclear metal complex (i.e.…”