NGEF Antibody, FITC conjugated

Definition and Structure

NGEF (Neuronal Guanine Nucleotide Exchange Factor) antibody conjugated with fluorescein isothiocyanate (FITC) is a fluorescently labeled immunoreagent designed for detecting NGEF proteins in cellular and tissue samples. FITC, a small organic dye, is covalently attached to the antibody’s primary amines (e.g., lysine residues) via a conjugation reaction. This labeling enables visualization under fluorescence microscopy or flow cytometry while retaining the antibody’s binding specificity for NGEF, a critical regulator of RHOA, RAC1, and CDC42 GTPases in neuronal signaling pathways .

Conjugation Process and Optimization

The conjugation protocol involves several key steps:

• Reaction Conditions: FITC reacts with antibody lysines in alkaline buffers (pH 9.2–9.5) at room temperature for 1–2 hours. Initial protein concentrations (25 mg/ml) and FITC-to-antibody ratios (10–400 µg FITC/mg antibody) are optimized to balance brightness and solubility .

• Purification: Excess FITC is removed via gel filtration or dialysis. Sodium azide, a common preservative in antibody buffers, must be excluded during conjugation to prevent interference .

• Labeling Index: Typical conjugation yields 3–6 FITC molecules per antibody. Higher indices risk quenching fluorescence and reducing binding affinity (Kd), as shown in studies where labeling density negatively correlated with antigen recognition .

Table 1: Key Conjugation Parameters

Applications in Research

The NGEF-FITC conjugate is employed in:

• Immunofluorescence (IF): Detecting NGEF localization in neurons or dendritic spines. Studies highlight its utility in axon guidance and spine morphogenesis research .

• Flow Cytometry (FC): Quantifying NGEF expression in cell populations, such as neurons or glial cells .

• Western Blotting (WB): While unconjugated NGEF antibodies are standard, FITC-conjugated variants enable fluorescence-based detection in multiplex assays .

Table 2: Applications and Performance Metrics

Research Insights

1. Binding Affinity: Studies reveal that higher FITC labeling indices correlate with reduced antigen binding affinity, underscoring the need for titration .

2. Spectral Properties: FITC emits at 525 nm (excitation: 490 nm), enabling compatibility with argon lasers and green fluorescent protein (GFP) imaging systems .

3. Cross-Reactivity: Polyclonal NGEF antibodies (e.g., ABIN6243778, 30229-1-AP) show reactivity across human, mouse, and rat tissues, with no reported cross-reactivity to non-target proteins .

Table 3: Spectral and Functional Properties