NGEF Antibody, HRP conjugated

Introduction to NGEF Antibody, HRP Conjugated

The NGEF (Neuronal Guanine Nucleotide Exchange Factor) antibody conjugated with Horseradish Peroxidase (HRP) represents a critical tool in immunoassays and biological research. NGEF is a key regulator of small GTPases (e.g., RHOA, RAC1, CDC42) involved in neuronal development, axon guidance, and dendritic spine morphogenesis . HRP conjugation enhances the antibody's utility in enzyme-linked immunosorbent assays (ELISA), immunohistochemistry (IHC), and Western blotting (WB) by enabling enzymatic signal amplification .

Structure and Function

Antibody Component:

• NGEF antibodies are typically polyclonal (rabbit IgG) or monoclonal, targeting epitopes such as the N-terminal region (AA 187-220) or C-terminal domains .

• Reactivity spans human, mouse, and rat samples, with validated applications in IHC (1:40-1:200 dilution), WB (1:1000-1:8000), and ELISA .

HRP Conjugation:

• HRP is a 44 kDa glycoprotein containing 6 lysine residues, which facilitate covalent binding to antibodies via periodate oxidation or maleimide-based chemistries .

• Conjugated antibodies retain enzymatic activity, enabling chromogenic (e.g., DAB), chemiluminescent (e.g., ECL), or fluorogenic substrate reactions .

Production and Optimization

HRP-conjugated NGEF antibodies are synthesized through:

1. Periodate Oxidation: Activates HRP carbohydrates to form aldehyde groups, which react with antibody lysines .

2. Lyophilization: Enhances conjugation efficiency by reducing reaction volume, yielding higher HRP:antibody ratios (e.g., 4:1) .

3. Affinity Purification: Ensures specificity and minimizes cross-reactivity .

Recombinant methods (e.g., Pichia pastoris expression) have also been explored to produce HRP-antibody conjugates with improved stability .

Applications in Research

Neuroscience:

• Analyzes axon guidance and dendritic spine morphogenesis via Ephrin-EPHA4 signaling .

• Detects NGEF in brain tissue (human, mouse, rat) via IHC .

Cancer and Immune Studies:

• Investigates NGEF's role in cell migration and adhesion .

• Monitors therapeutic antibodies (e.g., tanezumab) targeting NGF signaling .

Diagnostic Assays:

• ELISA for NGEF quantification in serum/plasma (detection limit: 1.5 ng/mL) .

• Multiplex platforms (e.g., Luminex) for high-throughput analysis .

Performance Metrics