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A rapid and cost-effective fluorescence detection in tube (FDIT) method to analyze protein phosphorylation.
- Source :
-
Plant Methods . 11/3/2016, Vol. 12, p1-6. 6p. - Publication Year :
- 2016
-
Abstract
- Background: Protein phosphorylation is one of the most important post-translational modifications catalyzed by protein kinases in living organisms. The advance of genome sequencing provided the information of protein kinase families in many organisms, including both model and non-model plants. The development of proteomics technologies also enabled scientists to efficiently reveal a large number of protein phosphorylations of an organism. However, kinases and phosphorylation targets are still to be connected to illustrate the complicated network in life. Results: Here we adapted Pro-Q® Diamond (Pro-Q® Diamond Phosphoprotein Gel Stain), a widely used phosphoprotein gel-staining fluorescence dye, to establish a rapid, economical and non-radioactive fluorescence detection in tube (FDIT) method to analyze phosphorylated proteins. Taking advantages of high sensitivity and specificity of Pro-Q® diamond, the FDIT method is also demonstrated to be rapid and reliable, with a suitable linear range for in vitro protein phosphorylation. A significant and satisfactory protein kinase reaction was detected as fast as 15 min from Wheat Kinase START 1.1 (WKS1.1) on a thylakoid ascorbate peroxidase (tAPX), an established phosphorylation target in our earlier study. Conclusion: The FDIT method saves up to 95% of the dye consumed in a gel staining method. The FDIT method is remarkably quick, highly reproducible, unambiguous and capable to be scaled up to dozens of samples. The FDIT method could serve as a simple and sensitive alternative procedure to determine protein kinase reactions with zero radiation exposure, as a supplementation to other widely used radioactive and in-gel assays. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 17464811
- Volume :
- 12
- Database :
- Academic Search Index
- Journal :
- Plant Methods
- Publication Type :
- Academic Journal
- Accession number :
- 119453277
- Full Text :
- https://doi.org/10.1186/s13007-016-0143-5