A rapid and cost-effective fluorescence detection in tube (FDIT) method to analyze protein phosphorylation. Issue 1 (December 2016)

• Record Type: Journal Article
• Authors: Jin, Xiao; Gou, Jin-Ying
• Is Part Of: Plant methods. Volume 12:Issue 1(2016)
• Page Start: 1
• Page End: 6
• Publication Date: 2016-12
• Subjects: Protein kinase reaction -- Fluorescence -- Pro-Q® diamond -- Wheat Kinase START 1 -- Thylakoid ascorbate peroxidase; Botany -- Methodology -- Periodicals; 572.2
• DOI: 10.1186/s13007-016-0143-5
• Languages: English
• ISSNs: 1746-4811

Abstract

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.