N-(m-PEG4)-N'-(azide-PEG3)-Cy5 | CAS 2107273-02-7

N-(m-PEG4)-N'-(azide-PEG3)-Cy5 is a Cy5-based fluorescent labeling building block bearing an azide-PEG3 handle for bioorthogonal click chemistry and an mPEG4 spacer to improve aqueous compatibility and reduce nonspecific interactions. Researchers use this reagent to generate fluorescent conjugates for imaging and quantification workflows where a reactive azide enables efficient coupling to complementary cyclooctyne/DBCO-functional partners. The Cy5 chromophore provides red spectral readout that is commonly integrated into fluorescence microscopy, plate-based assays, and flow cytometry experiments.

1. Azide Click Labeling

N-(m-PEG4)-N'-(azide-PEG3)-Cy5 is used as an azide-functional fluorescent tag for constructing fluorescent bioconjugates via strain-promoted azide-alkyne cycloaddition (SPAAC) with cyclooctyne/DBCO-bearing biomolecules. Molecular imaging and chemical biology groups incorporate the Cy5 signal to track conjugate formation, monitor labeling efficiency, and visualize labeled targets in cell-based or biomaterial-associated systems. The PEG spacers help present the Cy5 fluorophore away from the conjugation locus, supporting more reproducible fluorescence behavior in conjugates and probe libraries.

2. Fluorescence Microscopy Tracing

N-(m-PEG4)-N'-(azide-PEG3)-Cy5 supports fluorescence microscopy experiments where a reactive azide is needed to attach a Cy5 reporter to proteins, peptides, or other targeting ligands. Cell biology and microscopy teams use the resulting click-labeled conjugates to study binding, uptake, and localization patterns on cells or in extracellular matrices, including workflows where post-labeling with a click handle is preferred over pre-functionalized dyes. The red Cy5 emission is particularly convenient for multicolor imaging panels that include green or blue channels, enabling clear spatial readouts of labeled constructs.

3. Flow Cytometry Conjugates

N-(m-PEG4)-N'-(azide-PEG3)-Cy5 is frequently employed to prepare Cy5-labeled affinity reagents or labeling reagents for flow cytometry readouts after click coupling to DBCO-functional binders. Immunology and cell-surface profiling workflows benefit from the azide handle because it allows modular assembly of fluorescent conjugates from a common Cy5-azide reagent and a chosen targeting partner. The PEGylated format helps maintain conjugate dispersibility in aqueous buffers, supporting consistent staining and signal collection across experimental batches.

4. Plate-Based Fluorescence Assays

N-(m-PEG4)-N'-(azide-PEG3)-Cy5 is used in fluorescence assay development where a Cy5 reporter is required on a click-assembled probe, such as affinity reagents, binding standards, or fluorescent readout components for screening. Assay developers leverage the azide functionality to generate labeled constructs on demand, facilitating rapid optimization of probe architecture while keeping the fluorescence readout in the red channel. This reagent is also used to establish calibration or reference materials for comparing fluorescence responses of click-labeled conjugates across assay plates.