Detection of metabolic labeling with azido-sugars in different cell types using azide-reactive DyLight DyesA549, U2OS and HK-2 cells were incubated with 40µM azido-acetylmannosamine in cell culture media for 72 hours and then incubated with 100µM of DyLight 550-Phosphine (yellow). The cells were washed, fixed with 4% paraformaldehyde and counterstained with Hoechst 33342 (blue). Comparison of fluorescent detection reagents for metabolically labeled sugarsCell extracts were prepared from the A549 incubated with azido-sugarN-azidoacetylgalactosamine (ManNAz),N-azidoacetylglucosamine (GlcNAz) orN-azidoacetylmannosamine (GalNAz). The cell extracts were incubated with either Thermo Scientific DyLight 650-Phosphine or with Click-iT Alexa Fluor 647 DIBO Alkyne and analyzed by SDS-PAGE. The samples incubated with DyLight 650-Phosphine show a different labeling pattern for each of the three incorporated azido-sugar, demonstrating that different types of glycosylation (i.e., one sugar vs. another) can be detected specifically by using the DyLight Phosphine-activated detection reagents. Chemical structures of Azido-Sugars for metabolic labelingAll three compounds have the same molecular weight (MW 430.37). Once these compounds are incorporated into molecular structures, they can be detected using phosphine-activated molecules via the Staudinger ligation reaction chemistry. |
QQ:1749072012