Thermo Scientific DyLight Fluor Phosphine Reagents are phosphine-activated fluorescent dyes for specific labeling and detection of azide-tagged molecules, which enables use of fluorescence imaging in metabolic labeling strategies.
When used in combination with azide labeling strategies, phosphine-activated DyLight Fluors enable selective fluorescent labeling for detection of protein interactions and post-translational modifications using fluorescence imaging technologies. The phosphine group conjugates to azide groups by the Staudinger reaction mechanism. Azide groups can be introduced into proteins or other cellular targets through in vivo labeling with azide-derivatives of naturally occurring metabolic building blocks (bioorthogonal compounds). Because neither phosphines nor azides are present in biological systems, they comprise a chemoselective (mutually specific) ligation pair for labeling and conjugation.
Highlights:
- Soluble – easily dissolves in water-miscible solvents (e.g., DMSO) for subsequent dilution in aqueous reaction mixtures with cell lysates and other biological samples
- Compatible – reaction chemistry occurs effectively in simple buffer conditions; requires no accessory reagents such as copper or reducing agents, and does not interfere with fluorescence applications
- Chemoselective – the phosphine reactive group is specific in biological samples for azide-tagged molecules, ensuring that fluorescent labeling is specific
- High-performance fluorescence – DyLight 488, 550 and 650 are intense, highly stable fluorophores for green, orange and red fluorescent detection. (see DyLight Fluors)
Product Details:
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Overview of Staudinger ligation (azide-phosphine conjugation). Phosphine-activated compounds conjugate with high specificity to azide-tagged molecules, resulting in stable covalent attachment of "A" and "B" molecules. For additional information, see our review of Staudinger ligation reaction chemistry.
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In vivo detection of metabolically incorporated azido-acetylgalactosamine using Thermo Scientific DyLight 550- and 650-Phosphine Labeling Reagents. Panel A. U2OS cells were incubated with 40µM azido-acetylgalactosamine in cell culture media for 72 hours and the live cells were incubated with 100µM of DyLight 550-phosphine . The cells were then washed, fixed with 4% paraformaldehyde and counterstained with Hoechst 33342. (green: DyLight 550-labeled azido-galactosamine, blue: Hoechst 33342 labeled nuclei). Panel B. HK-2 cells were incubated with 40µM azido-acetylmannosamine in cell culture media for 72 hours and the live cells were incubated with 100µM of DyLight 650-phosphine. The cells were then washed, fixed with 4% paraformaldehyde and counterstained with Hoechst 33342. (cyan: DyLight 650-labeled azido-mannosamine, red: Hoechst 33342 labeled nuclei).
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| Detection of metabolically incorporated azido-sugars on live and fixed cells using Thermo Scientific DyLight DyLight 550- and 650-Phosphine Labeling Reagents. HK-2 cells were incubated with 40µM azido-acetylmannosamine in cell culture media for 72 hours. The azido-sugar were labeled either after 4% paraformaldehyde fixation using DyLight 550-phoshine (Panel A) or labeled in live cells with DyLight 650-Phosphine Labeling Reagent (Panel B). The cells were washed and counterstained with Hoechst 33342. Panel A. The golgi structure (green) is predominantly detected by fixed cell labeling, where as the cell membrane and secretory vesicles (red) are labeled by live-cell labeling (blue: Hoechst 33342 labeled nuclei). |
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