A Laborsaving, Timesaving, and More Reliable Strategy for Separation of Low-Molecular-Mass Phosphoproteins in Phos-tag Affinity Electrophoresis

Abstract

Reversible phosphorylation is a key signaling mechanism for modulating the functional properties of proteins involved in numerous cellular events. Abnormal protein phosphorylation causes many human diseases. Experimental procedures for the determination of the phosphorylation status of certain proteins are therefore very important in relation to studies on a diverse range of physiological and pathological processes. We have previously reported a novel phosphate-affinity sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technique using a dizinc(II) complex of the phosphate-binding ligand Phos-tag in conjunction with a neutral-pH gel system to detect shifts in the mobilities of phosphoproteins (Zn 2+ -Phos-tag SDS-PAGE). However, this handmade gel-based procedure is often laborious and time-consuming to perform, and requires a skillful analyst. More recently, SuperSep Phos-tag precast gel has been developed on the basis of the Zn 2+ -Phos-tag SDS-PAGE methodology. This novel ready-to-use system employs a neutral-pH gel containing 12.5% (w/v) polyacrylamide and the immobilized Zn 2+ -Phos-tag (50 µM), which is generally used in conjunction with a Tris-glycine-based electrophoretic running buffer. We examined the potential usage of a Tris-N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine (Tris-Tricine) buffer as an alternative running buffer for the SuperSep Phos-tag precast gel system in the analysis of low-molecular-mass phosphoproteins. Compared with Tris-glycine, the Tris-Tricine running buffer improved the resolution of 8.8-35 kDa phosphoproteins and phosphopeptides. We can therefore provide a laborsaving, timesaving, and more reliable strategy for separation of low-molecular-mass phosphoproteins in Phos-tag affinity electrophoresis.

Oncogenic mutant RAS signaling activity is rescaled by the ERK/MAPK pathway

“…We employed the Phos-Tag method using precast gels (SuperSep Phos-Tag 12.5% Cat #195-17991, and 7.5% Cat #192-18001, FUJIFILM Wako Chemicals). However, in accordance with previous observations with these gels (Kinoshita-Kikuta et al, 2012) and the observation that they likely have excess Phos-Tag reagent, we use samples collected in EDTA-containing RIPA buffer and we perform electrophoresis with Tris-Glycine running buffer (as with above). Phos-Tag fractional phosphorylation measurements are internally controlled and required neither cross-load or cross-blot normalization.…”

Advances in Phos-tag-based methodologies for separation and detection of the phosphoproteome

“…Recently, the Zn 2+ -Phos-tag SDS-PAGE precast gels that we developed have been commercialized by Wako Pure Chemical Industries, Ltd. as SuperSep Phos-tag. We used this product, with a gel concentration (w/v) of 12.5% and a Phos-tag concentration of 50 μM, to demonstrate an example of the separation and detection of the phosphorylation status of histone H3, an intracellular protein [39].…”

The Cutting Edge of Affinity Electrophoresis Technology

“…The neutral Phos-tag SDS-PAGE is more durable, and it permits the development of a precast gel with an assured long-term quality and shelf life. Recently, the neutral Phos-tag SDS-PAGE precast gels have been commercialized by Wako Pure Chemical Industries, Ltd. (Osaka, Japan) as SuperSep Phos-tag [ 33 ]. Use of the precast gel is worthy of consideration in terms of labor saving, time saving and reliability in the detection of phosphoproteins.…”

“…This article describes advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years. In addition, it introduces a technique for the analysis of phosphorylated proteins that is based on the phosphate-affinity probe, Phos-tag [ 11 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 ], which we developed, and it describes the contributions made to phosphoproteomics by techniques based on Phos-tag.…”