Flaas

GST-MoCka1, GST-MoCkb1, GST-MoCkb2, His-MoRgs1, and His-MoRgs1^5A were expressed in E. coli DE3 cells and purified. A rapid and cost-effective fluorescence detection in tube (FDIT) method was used to analyze in vitro protein phosphorylation [74 (link)]. The Pro-Q Diamond Phosphorylation Gel Stain (Thermo Fisher Scientific, P33301) is used for phosphor-protein gel-staining. For protein kinase reactions, 2 μg MoRgs1 (MoRgs1^5A) was mixed with MoCka1, MoCkb1, and MoCkb2 in a kinase reaction buffer (100 mM PBS [Beyotime Biotechnology, ST476], pH 7.5, 10 mM MgCl₂, 1 mM ascorbic acid [Sigma-Aldrich, A5960]), with the appearance of 50 μM ATP (Sigma-Aldrich, FLAAS) at room temperature (RT) for 60 min, 10 folds of cold acetone was added to stop the reaction. For protein in tube staining, samples were homogenized and suspended in Mili-Q water at the concentration of 0.2 μg/μl. The pellet was rinsed with 0.5 ml cold acetone and centrifuge to remove the supernatant twice. The pellet was air-dried and dissolved in 200 μl of Mili-Q water and moved to a black 96 well plate (Corning, 3925). Fluorescence signal at 590 nm (excited at 530 nm) was measured in a Cytation3 microplate reader (Biotek, Winooski, VT, USA) [73 (link)].

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)].