Rapid Fluorescence Detection of Protein Phosphorylation

A rapid and cost-effective fluorescence detection in tube (FDIT) method using phosphoprotein gel stain was used for in vitro phosphorylation analysis [33 (link),34 (link),57 (link)]. Proteins GST-MoMkk1, His-MoAtg4, His-MoAtg3, GST-MoAtg5, and His-MoAtg16 were expressed in E. coli BL21 cells and purified using anti-GST or anti-His beads. The following reagents were placed in a 1.5 ml centrifuge tube: 0.2 μg kinase GST-MoMkk1, 2 μg substrate His-MoAtg4, His-MoAtg3, GST-MoAtg5, or His-MoAtg16 with ATP at a final concentration of 50 μM (Sigma-Aldrich, FLAAS), and sufficient kinase reaction buffer (100 mM PBS, 10 mM MgCl₂, 1 mM ascorbic acid, pH 7.5) to 100 μl volume for phosphorylation reaction at room temperature for 1 h, then 10-fold of cold acetone was added to stop the reaction. Phosphorylation protein was stained by Pro-Q Diamond Phosphorylation Gel Stain (Thermo Fisher Scientific, P33301), the widely used phosphor-protein gel-staining fluorescence dye, in the dark at room temperature for 1 h. Proteins were pelleted with cold acetone and washed twice with 0.5 ml of cold acetone. The protein pellet then was dissolved in 200 μl of ddH₂O after drying and measured phospho-fluorescence at 590 nm (excited at 530 nm) using a Cytation3 microplate reader (Biotek, Winooski, VT, USA). Groups without kinase or substrate proteins were set up as controls [14 (link)].