Bis-(N,N'-PEG4-NHS ester)-Cy5 | CAS 2107273-48-1

Bis-(N,N'-PEG4-NHS ester)-Cy5 is a Cy5-based fluorescent labeling reagent featuring two N-hydroxysuccinimide (NHS) ester groups on PEG4 linkers, enabling efficient conjugation to primary amines on proteins, peptides, and other amine-containing biomolecules. The Cy5 fluorophore is designed for far-red fluorescence imaging workflows, where bright labeling and reduced background from spectral separation are often advantageous for microscopy and fluorescence-based assays.

1. Protein Conjugation Labeling

Bis-(N,N'-PEG4-NHS ester)-Cy5 is used to generate far-red fluorescent protein conjugates for imaging and quantitative fluorescence readouts. Researchers commonly apply it to label antibodies, antibody fragments, enzymes, and purified protein standards where primary amines are available on lysine residues or N-termini. The dual NHS-ester architecture supports multivalent attachment patterns that can increase effective dye loading on the protein scaffold, while the PEG4 spacers help maintain labeling flexibility and reduce steric constraints that can otherwise affect binding or activity in downstream assays.

2. Antibody and Affinity Reagent Imaging

Bis-(N,N'-PEG4-NHS ester)-Cy5 is frequently incorporated into labeled affinity reagents for cellular and biomolecular localization studies. Fluorescent antibody conjugates prepared with this reagent are used for immunofluorescence microscopy and fluorescence-based binding experiments, including workflows where far-red Cy5 emission helps distinguish specific signal from autofluorescence. The PEGylated, amine-reactive labeling strategy supports preparation of imaging reagents for target-binding experiments, flow cytometry panels, and multicolor experiments where careful spectral spacing is required.

3. Flow Cytometry Fluorescent Tagging

Bis-(N,N'-PEG4-NHS ester)-Cy5 supports fluorescent labeling strategies for flow cytometry when amine-containing targeting molecules are available. Investigators use Cy5-labeled conjugates to track binding of antibodies, ligands, or other protein reagents to cell-surface or intracellular targets after appropriate sample preparation. The far-red emission of Cy5 is commonly leveraged to improve separation from shorter-wavelength fluorophores and cellular background, enabling robust gating and quantification of labeled populations in fluorescence cytometry workflows.

4. Biomaterial Surface Functionalization

Bis-(N,N'-PEG4-NHS ester)-Cy5 is applied in biomaterials research to create fluorescently tagged surfaces and coatings for microscopy and material characterization. The NHS ester functionality enables coupling to amine-bearing surfaces such as amine-functional polymers, modified glass/metal oxides, or biomaterial layers containing accessible primary amines. PEG4 spacing can help preserve surface accessibility of the dye while providing a more uniform labeling distribution for visualization of coatings, tracking of material interfaces, and qualitative assessment of surface coverage in imaging experiments.