ATTO 514 NHS ester

ATTO 514 NHS ester is a reactive fluorescent dye designed for efficient covalent labeling of primary amines via NHS ester chemistry. As a bright ATTO 514 fluorophore, it is commonly used in fluorescence microscopy and flow cytometry workflows where robust dye conjugation is required. Its amine-reactive functionality makes it a practical reagent for preparing fluorescent conjugates from proteins, peptides, and other biomolecules bearing lysine residues or engineered amine groups.

1. Protein And Antibody Labeling

ATTO 514 NHS ester is widely used to generate fluorescent protein conjugates for imaging and quantitative fluorescence assays, including antibody labeling for immunostaining workflows. Researchers typically react the NHS ester with lysine-containing antibodies, enzymes, or affinity reagents to produce stable amine-linked dye conjugates that can be tracked by fluorescence readout in microscopy, plate-based assays, or instrument-based detection. This labeling approach is especially useful when a defined dye-to-biomolecule conjugate is needed for consistent signal in comparative experiments.

2. Fluorescence Microscopy Staining

ATTO 514 NHS ester supports fluorescence microscopy staining of fixed cells and biomolecular targets by enabling covalent attachment of the dye to amine-rich cellular components or to antibody/affinity binders used for immunofluorescence. In imaging workflows, the NHS ester format helps researchers incorporate ATTO 514 into staining reagents that can be visualized on standard fluorescence microscopes using the dye's visible excitation/emission window. The covalent labeling strategy is also used when wash resistance is important for multi-step staining protocols and for reducing dye loss during imaging.

3. Flow Cytometry Conjugates

ATTO 514 NHS ester is frequently used to prepare flow cytometry dyes by covalently labeling amine-bearing proteins, antibodies, or other targeting ligands used for cell-surface or intracellular labeling strategies. In cytometry workflows, the dye conjugate enables fluorescence-based gating and population analysis with a stable covalent attachment that tolerates typical staining and washing steps. This reagent format is particularly relevant for multiplex panel development where consistent conjugation chemistry helps maintain reproducible fluorescence across samples and experimental replicates.

4. Fluorescent Standards And Tracers

ATTO 514 NHS ester is also used in reagent development to create fluorescent standards, calibration materials, and tracer conjugates for assay and imaging method optimization. By labeling amine-containing reference proteins or polymers, researchers can generate reproducible fluorescent materials for instrument setup, background checking, and workflow validation. The covalent NHS ester chemistry provides a straightforward route to stable fluorescent tracers that remain associated with the labeled scaffold during handling, dilution, and measurement.