Premium
A rapid and simple 8‐quinolinol‐based fluorescent stain of phosphoproteins in polyacrylamide gel after electrophoresis
Author(s) -
Wang Xu,
Hwang SunYoung,
Cong WeiTao,
Jin LiTai,
Choi JungKap
Publication year - 2015
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201500249
Subject(s) - fluorescence , phosphoprotein , chromatography , chemistry , stain , chelation , phosphoproteomics , polyacrylamide gel electrophoresis , ultraviolet light , capillary electrophoresis , electrophoresis , casein , staining , biochemistry , biology , kinase , phosphorylation , protein phosphorylation , physics , protein kinase a , organic chemistry , photochemistry , quantum mechanics , genetics , enzyme
In order to obtain an easy and rapid protocol to visualize phosphoproteins in SDS‐PAGE, a fluorescent detection method named 8‐Quinolinol (8‐Q) stain is described. 8‐Q can form ternary complexes in the gel matrix contributed by the affinity of aluminum ion (Al 3+ ) to the phosphate groups on the proteins and the metal chelating property of 8‐Quinolinol, exhibiting strong fluorescence in ultraviolet light. It can visualize as little as 4∼8 ng of α‐casein and β‐casein, 16∼32 ng of ovalbumin and κ‐casein which is more sensitive than Stains‐All but less sensitive than Pro‐Q Diamond. The protocol of 8‐Q requires only 70 min in 0.75 mm mini‐size or 1.0 mm large‐size gels with five changes of solutions without destaining step; Pro‐Q takes at least 250 min with 11 changes of solutions. In addition, the new method was confirmed by the study of dephosphorylation and LC‐MS/MS, respectively. The approach to visualize phosphoprotein utilizing 8‐Q could be an alternative to simplify the analytical operations for phosphoproteomics research.