A rapid and cost-effective fluorescence detection in tube (FDIT) method to analyze protein phosphorylation
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Plant Methods Open Access
METHODOLOGY
A rapid and cost‑effective fluorescence detection in tube (FDIT) method to analyze protein phosphorylation Xiao Jin and Jin‑Ying Gou*
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 technolo‑ gies 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 phospho‑ protein 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 ProQ® 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. Keywords: Protein kinase reaction, Fluorescence, Pro-Q® diamond, Wheat Kinase START 1, Thylakoid ascorbate peroxidase Background During evolution, living organisms developed accurate and strict cellular regulatory systems to control all biological aspects in a cell from gene transcription to protein post-translational modifications (PTMs). Protein phosphorylation is the most abundant and widespread PTM, and represents over 50% of PTMs, although more than 300 different types of PTMs have been reported (http://www.abrf.org). Tightly regulated and rapid reversible protein phosphorylation affords organisms the *Correspondence: [email protected] 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
advantages to survive, via adding or removing a phosphoryl group to targets catalyzed by protein kinases or phosphatases through consuming ATP, the most commonly used cofactor in life, which saves both time and energy [1]. At the protein level, phosphorylation is critical for the structural and functional state of proteins to funct
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