AF 555 NHS ester

AF 555 NHS ester is an NHS-ester activated fluorescent dye designed for covalent fluorescent labeling of primary amine-containing biomolecules. This reactive ester enables efficient coupling under standard bioconjugation conditions to generate stable amide-linked conjugates for fluorescence imaging and fluorescence-based assays. With excitation and emission in the orange-red range, AF 555 conjugates are frequently used when bright, spectrally separated labeling is needed for microscopy and flow cytometry workflows.

1. Protein And Antibody Labeling

AF 555 NHS ester is widely used by protein bioconjugation teams to fluorescently tag antibodies, antibody fragments, enzymes, and carrier proteins for downstream immunofluorescence and binding studies. Researchers rely on the NHS-ester chemistry to attach the dye to lysine residues on proteins, producing amine-reactive conjugates that can be incorporated into staining panels and fluorescence imaging experiments. In microscopy and flow cytometry workflows, AF 555-labeled binders help visualize target localization and quantify binding readouts using standard fluorescence channels compatible with orange-red emission detection.

2. Fluorescence Microscopy Staining

AF 555 NHS ester supports fluorescence microscopy staining of fixed cells and biomolecular samples where covalent labeling provides robust signal retention during washing and imaging. Typical use cases include labeling extracellular proteins, extracellular matrix components, or surface-associated targets by preparing AF 555-conjugated reagents prior to imaging. Imaging groups also use AF 555 conjugates to build multicolor staining schemes, leveraging its orange-red spectral position to reduce spectral overlap with common green and far-red fluorophores when designing microscopy experiments and colocalization studies.

3. Flow Cytometry Conjugate Preparation

AF 555 NHS ester is commonly used in flow cytometry reagent development to prepare fluorescently labeled antibodies and protein probes for cell-surface and intracellular staining workflows. Cytometry users generate AF 555 conjugates that can be titrated to match instrument settings and used to quantify fluorescent intensity distributions by gating strategies. Because NHS-ester labeling yields covalent amide linkages to lysine-rich proteins, AF 555-tagged reagents are frequently selected when consistent labeling and wash-resistant fluorescence are important for comparing sample populations across experimental replicates.

4. Fluorescent Bioassay Reagent Development

AF 555 NHS ester is also applied in fluorescence assay development where labeled proteins or labeling standards are required for readouts such as binding assays, fluorescence-based quantification, and assay calibration controls. Researchers prepare AF 555-conjugated biomolecules to serve as fluorescent reporters in plate-based experiments, including conjugate-based detection formats and fluorescence tracking of biomolecular interactions. In these settings, the dye's stable covalent attachment via NHS-ester coupling helps maintain labeling integrity during assay incubation and washing steps, supporting reproducible fluorescence measurements.