A mixture of 50 or 100 ?M Azido-Cy5/FAM (10 mM stock solution in DMSO); 1 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (50 mM freshly prepared stock solution in ddH 2 O); 0.2 mM tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) (2 mM stock in a mixture of tertbutanol and DMSO (4:1)); 1 mM CuSO 4 -5H 2 O (50 mM freshly prepared stock in ddH 2 O) was added to the 20 µL Lnt reaction solutions and samples were incubated at room temperature for 1 h. After incubation, an aliquot of each sample was diluted in loading buffer and heated at 100 °C for 5 min and loaded onto home-made Tris-Tricine SDS-PAGE gels for separation of peptides or processed for detection by fluorescence spectroscopy, vol.9, p.15978, 2019. ,
Peptides were separated in high-resolution ,
, Urea SDS-PAGE gels consisting of three sequentially polymerized layers: (i) resolving gel 20% containing 6 M urea; (ii) spacer gel 11% with 4 M urea and (iii) stacking gel 4% 18 . Gels migrated at room temperature in a Mini-PROTEAN ® Tetra Vertical Electrophoresis Cell
, Bio-Rad) and hybridized against streptavidin peroxidase (HRP) conjugate (for FSL-1-biotin) or directly analyzed by fluorescent detection (for FSL-1-fluorescein). SuperSignal ? West Femto Maximum Sensitivity Substrate Western blotting detection reagent (Thermo Scientific) was used as substrate. Chemiluminescent and fluorescent signals were detected with a Chemidoc MP gel imaging system, FSL-1-biotin or FSL-1-fluorescein bands
, Monitoring product formation in 96-well plate format by fluorescence spectroscopy
, Click chemistry samples containing the biotin-conjugated peptide FSL-1 (20 ?L) were diluted in PBST1 (80 ?L) and added to each well of a 96-well plate. The plate was incubated at room temperature while shaking for 1 h in the dark to bind biotin-conjugates onto the streptavidin-coated surface, after which the wells were rinsed six times with PBST2 (PBS with 1% Tween-20), three times with PBST1 (0.05% Tween20) and three times with PBS, Home-made streptavidin-coated microplate were obtained by incubation of 100 ?L of 10 ?g/mL streptavidin per well on 96-well plates (Greiner)
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