Alex fluor 488 maleimide

Flagellar filament (flaB1 T166C flaA1 T174C; 38) and hook (flgE1 T183C) structures of exponentially growing cells were fluorescently labeled using Alex Fluor 488 maleimide and Alex Fluor 568 maleimide (Thermo Fisher Scientific) in accordance with previously publicized methods (Bubendorfer et al., 2012 (link)).

MBP-SHLD2^1–60 containing C-terminal LPETGG motif was labeled using sortase A transpeptidase enzyme from Staphylococcus aureus. Labeling was carried out by mixing 100 μM MBP-SHLD2^1–60 with 10 μM Sortase A enzyme and 0.5–1 mM of AlexaFluor488-conjugate peptide (GlyGlyGlyCys-AlexaFluor488). The conjugation of AlexaFluor488 to the peptide was carried out by incubating GGGC peptide (GenScript) with AlexFluor488-maleimide (Thermo Fisher Scientific) in 100 mM HEPES-NaOH pH 7.4 at equimolar ratio at 25 °C for 2 h. The labeling reaction was carried out with overnight incubation in buffer B supplemented with 10 mM CaCl₂ at 4 °C. Labeled MBP-SHLD2^1–60 was separated from excess AlexaFLuor488-conjugate peptide by SEC (Superdex-75, Cytiva).