Fluorescein-PEG5-NHS ester | CAS 2353409-62-6

Fluorescein-PEG5-NHS ester is a fluorescein-based labeling reagent featuring an NHS ester for efficient covalent attachment to primary amines on proteins, peptides, and other amine-containing biomolecules. The PEG spacer helps improve aqueous handling and labeling performance by reducing steric constraints during conjugation, making it a widely used fluorescent tag for constructing research-grade fluorescent conjugates. Its fluorescein emission is commonly leveraged for fluorescence microscopy and fluorescence-based assays where bright, visible-green readouts are advantageous.

1. Protein Fluorescent Labeling

Fluorescein-PEG5-NHS ester is frequently used by bioconjugation and protein engineering groups to generate fluorescently labeled antibodies, enzymes, and protein standards for imaging and binding studies. Researchers covalently attach the NHS-activated fluorescein to lysine residues or N-terminal amines to produce stable fluorescein conjugates that can be tracked in solution or on surfaces. The PEG5 linker is particularly helpful when labeling larger proteins or when maintaining accessibility of functional domains is important for downstream binding assays or immobilized-protein workflows.

2. Fluorescence Microscopy Tracing

Fluorescein-PEG5-NHS ester supports fluorescence microscopy workflows where amine-containing targets need to be visualized with a green-emitting dye. Typical use cases include labeling extracellular matrix components, cell-surface proteins, or tagged biomolecular carriers prior to imaging, enabling localization and trafficking studies in microscopy experiments. Because the reagent forms covalent conjugates through amine coupling, it is often chosen for experiments requiring retention of fluorescence signal during washes and multi-step staining or incubation protocols.

3. Flow Cytometry Conjugate Preparation

Fluorescein-PEG5-NHS ester is used in flow cytometry reagent development to prepare fluorescent conjugates for labeling cells or cell-associated biomolecules via amine coupling. Many laboratories generate fluorescein-labeled probes by reacting the NHS ester with amine-bearing ligands, detection reagents, or protein conjugates that can then be used in staining panels. The PEG spacer can reduce steric effects during conjugate formation, which is helpful when preparing labeled reagents intended to maintain binding behavior in flow-based readouts.

4. Fluorescence-Based Bioassays

Fluorescein-PEG5-NHS ester is commonly incorporated into fluorescence assay development where amine-reactive fluorescent tags are required for monitoring binding, uptake, or reaction progress. Researchers prepare labeled assay components such as fluorescent protein conjugates, labeled affinity reagents, or fluorescent standards that can be quantified using plate readers or benchtop fluorescence instruments. The fluorescein label provides a convenient optical reporter for many in vitro assay formats that rely on tracking fluorescence intensity changes associated with conjugate formation or target binding.