A rapid and simple 8-quinolinol-based fluorescent stain of phosphoproteins in polyacrylamide gel after electrophoresis

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 (Al3+) to the phosphate groups on the prote...

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Bibliographic Details
Published in:Electrophoresis 2015-10, Vol.36 (20), p.2522-2529
Main Authors: Wang, Xu, Hwang, Sun-Young, Cong, Wei-Tao, Jin, Li-Tai, Choi, Jung-Kap
Format: Article
Language:English
Subjects:
8-Quinolinol
Affinity
Aluminum
Aluminum ion
Diamonds
Electrophoresis
Electrophoresis, Polyacrylamide Gel - methods
Fluorescence
Fluorescent detection
Fluorescent Dyes - chemistry
Mass Spectrometry
Mathematical analysis
Oxyquinoline - chemistry
Phosphates
Phosphoprotein stain
Phosphoproteins - analysis
Phosphoproteins - chemistry
Polyacrylamides
Reproducibility of Results
SDS-PAGE
Sensitivity and Specificity
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Summary: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 (Al3+) 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.
ISSN:0173-0835
1522-2683
DOI:10.1002/elps.201500249