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 (Zn2+–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 Zn2+–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 Zn2+–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.
Highlights
Separation of a phosphorylated protein and its nonphosphorylated counterpart by using gel-based electrophoresis can facilitate an understanding of the phosphorylation status
There was only a slight difference in the migration patterns of the phosphorylated and the dephosphorylated forms in the Tris–glycine and Tris–morpholin-4-ylpropane-1-sulfonic acid (MOPS) systems. These results show that the use of Tris–Tricine as the running buffer permits greater resolution in separation analyses of standard phosphoproteins than that achieved by using the Tris–glycine buffer
We could not measure accurately their comparative electrophoretic mobilities in the Tris–glycine system. These results suggest that a combination of SuperSep Phos-tag with Tris–Tricine running buffer provides a higher coverage for a number of phosphoproteins/phosphopeptides and that it generates more-detailed information on the phosphorylation status of proteins/peptides in the low-molecular-mass range
Summary
Separation of a phosphorylated protein and its nonphosphorylated counterpart by using gel-based electrophoresis can facilitate an understanding of the phosphorylation status. If there is a sufficient decrease in charge density of the phosphorylated form, the phosphoprotein will show a retarded migration and will appear at a position corresponding to a higher apparent molecular weight on the gel compared with its nonphosphorylated counterpart. This observation of a shift in mobility has sometimes been used as an index of protein phosphorylation in certain biological events; the shift in mobility on phosphorylation depends on protein-specific structural characteristics, and the number of phosphoproteins that can be analyzed by the conventional SDS-PAGE technique is limited.
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