In Vitro Phosphorylation Assay of MoMkk1 by MoSep1

The His-MoSep1, GST-MoMkk1, GST-MoMkk1^S19A, GST-MoMkk1^T24A, GST-MoMkk1^S125A, GST-MoMkk1^S136A, GST-MoMkk1^T139A, GST-MoMkk1^T207A, GST-MoMkk1^6A were expressed in Escherichia coli BL21-CodonPlus (DE3) cells and purified. A rapid Fluorescence Detection in Tube (FDIT) method using Pro-Q Diamond Phosphorylation Gel Stain was used to analyze protein phosphorylation in vitro. First, 2 mg MoMkk1 (or the unphosphorylated mutations) was mixed with MoSep1 in a kinase reaction buffer (100 mM PBS, 1 mM ascorbic acid, pH 7.5, 10 mM MgCl₂), with 50 mM ATP at 25°C for 1 h. 10 folds of cold acetone was then added to terminate the reaction. Casein was homogenized and suspended in Mili-Q water at a concentration of 0.2 mg/ml to stain the proteins. Briefly, 100 ml of Pro-Q Diamond was mixed with 10 ml of casein and the mixture was kept in dark 1 h. 10 folds of cold acetone was then added and the mixture was allowed to incubate overnight at -20°C. Proteins were precipitated by centrifugation at 14,000 rpm for 1 h at 4°C. Discard the supernatant. The pellet (proteins) was washed twice using 500 μl cold acetone. The pellet was dissolved in 200 μl of Mili-Q water and transferred to a 96 well plate. Fluorescence signal at 590 nm (exited at 530 nm) was measured using a Cytation3 microplate reader.