A rapid and cost-effective fluorescence detection in tube (FDIT) method to analyze protein phosphorylation.
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.


CONCLUSIONS
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.
State Key Laboratory of Genetic Engineering, Institute of Plant Biology, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Fudan University, Shanghai, 200438 China.
Xiao
X
Jin
State Key Laboratory of Genetic Engineering, Institute of Plant Biology, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Fudan University, Shanghai, 200438 China.
Jin-Ying
JY
Gou
Fluorescence; Pro-Q® diamond; Protein kinase reaction; Thylakoid ascorbate peroxidase; Wheat Kinase START 1