Example 11
Purified bacteria sialidases (1 μg) were incubated with DFSA (0.1 mM) at room temperature for 1 h, and separated on 4-12% NuPAGE (Invitrogen). 5×105 influenza viruses were incubated with DFSA (0.1 mM) at room temperature for 1 h, and separated on 4-12% NuPAGE (Invitrogen). Sialidase transfectant 293T cells were lysed by different lysis buffers: pH4.5 (1% NP-40, 100 mM NaOAc, 150 mM NaCl, 3 mM KCl, pH 4.5, 1×EDTA-free protease inhibitor cocktail from Roche), pH 7.4 buffer (1% NP-40, 25 mM Tris, 150 mM NaCl, 3 mM KCl, pH 7.4, 1×EDTA-free protease inhibitor cocktail from Roche), and pH 9.0 buffer (1% NP-40, 25 mM Tris, 150 mM NaCl, 3 mM KCl, pH 9.0, 1×EDTA-free protease inhibitor cocktail from Roche). The lysates were collected and incubated with DFSA (0.1 mM) at 37° C. for 1 h. Following incubation, the samples were clarified, and protein concentrations were determined by bicinchoninic acid protein assay kit (Pierce). For each sample, 20 μg total lysate was separated on 4-12% NuPAGE (Invitrogen). After electrophoresis, the gels were blotted onto PVDF membranes (Millipore). Click reactions were performed on the PVDF membranes, and labeling signals were processed and analyzed by chemiluminescence detector.