Sequential double fluorescent detections of total proteins and phosphoproteins in SDS-PAGE

A fluorescent staining technique, using selective chelation with fluorophore and metal ion to the phosphate groups of phosphoproteins in SDS‐PAGE is described. As a fluorescent dye and a metal ion, Fura 2 pentapotassium salt and Al3+ were employed, respectively. The staining method, Fura 2 stain, ha...

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Bibliographic Details
Published in:Electrophoresis 2014-04, Vol.35 (8), p.1089-1098
Main Authors: Hwang, Sun-Young, Wang, Xu, Cong, Wei-Tao, Jin, Li-Tai, Choi, Jung-Kap
Format: Article
Language:English
Subjects:
Aluminum
Aluminum - analysis
Animals
Caseins - analysis
Cations - analysis
Cattle
Chelation
Chemical compounds
Dyes
Electrophoresis, Polyacrylamide Gel - methods
Fluorescence
Fluorescent detection
Fluorescent Dyes - analysis
Fura 2
Fura-2 - analysis
Horses
Low cost
Ovalbumin - analysis
Phosphates
Phosphoprotein
Phosphoproteins - analysis
Pro-Q Diamond
Proteins
Proteins - analysis
SDS-PAGE
Staining
Staining and Labeling - methods
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Summary:A fluorescent staining technique, using selective chelation with fluorophore and metal ion to the phosphate groups of phosphoproteins in SDS‐PAGE is described. As a fluorescent dye and a metal ion, Fura 2 pentapotassium salt and Al3+ were employed, respectively. The staining method, Fura 2 stain, has sensitivities of 16–32 ng of α‐casein and β‐casein, 62 ng of ovalbumin, phosvitin, and κ‐casein using an ultraviolet transilluminator. Furthermore, Fura 2 stain is able to carry out continuative double detection of total proteins and phosphoproteins on the same gel within 3.5 h. Consequently, selective phosphoprotein and total protein detections could be obtained without other poststaining. Considering the low cost, simplicity, and speed, Fura 2 staining may provide great practicalities in routine phosphoproteomics research.
ISSN:0173-0835
1522-2683
DOI:10.1002/elps.201300538