pro-q diamond gel stain for phosphorylation  (Thermo Fisher)

Journal: mBio

Article Title: MoMkk1 and MoAtg1 dichotomously regulating autophagy and pathogenicity through MoAtg9 phosphorylation in Magnaporthe oryzae

doi: 10.1128/mbio.03344-23

Figure Lengend Snippet: MoAtg9 is phosphorylated by MoMkk1. ( A ) In vivo phosphorylation analyses of MoAtg9-GFP proteins treated with phosphatase inhibitor (PI), phosphatase (PE), and detected by the anti-GFP antibody. ( B ) In vitro phosphorylation analysis by the fluorescence detection in tube (FDIT) method. Purified proteins of GST-MoMkk1 and His-MoAtg9 were used in protein kinase reactions in the presence of 50 µM ATP and then dyed with a Pro-Q Diamond Phosphorylation Gel Stain. The fluorescence signal at 590 nm (excited at 530 nm) was measured in a Cytation3 microplate reader. Error bars represent SD, and asterisks denote statistical significance ( P < 0.01).

Article Snippet: Phosphorylation protein was stained by 100 µL of Pro-Q Diamond Phosphorylation Gel Stain (Thermo Fisher Scientific, P33301) and kept in the dark at RT for 1 h. Then, the sample was added 10-fold of cold acetone and precipitated in a −20°C freezer for 4 h and centrifuged at 13,200 g for 1 h at 4°C again.

Techniques: In Vivo, In Vitro, Fluorescence, Purification, Staining