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

• 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
• ISSN: 0173-0835; 1522-2683
• DOI: 10.1002/elps.201500249

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.