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

Content Provider	Springer Nature : BioMed Central
Author	Jin, Xiao Gou, Jin-Ying
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.
Related Links	https://plantmethods.biomedcentral.com/counter/pdf/10.1186/s13007-016-0143-5.pdf
Ending Page	6
Page Count	6
Starting Page	1
File Format	HTM / HTML
ISSN	17464811
DOI	10.1186/s13007-016-0143-5
Journal	Plant Methods
Issue Number	1
Volume Number	12
Language	English
Publisher	BioMed Central
Publisher Date	2016-11-03
Access Restriction	Open
Subject Keyword	Plant Sciences Biological Techniques Protein kinase reaction Fluorescence Pro-Q® diamond Wheat Kinase START 1 Thylakoid ascorbate peroxidase
Content Type	Text
Resource Type	Article
Subject	Plant Science Biotechnology Genetics
Journal Impact Factor	4.7/2023
5-Year Journal Impact Factor	5.6/2023

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Thylakoid-bound ascorbate peroxidase in spinach chloroplasts and photoreduction of its primary oxidation product monodehydroascorbate radicals in thylakoids. Plant cell physiology 33: 541–553 (1992).

Correction: Rapid and cost-effective molecular karyotyping in wheat, barley, and their crossprogeny by chromosome-specific multiplex PCR

A protein phosphorylation threshold for functional stacking of plant photosynthetic membranes (2010).

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