ADGRG3 Antibody, FITC conjugated

Molecular Basis

ADGRG3 (GPR97) is a member of the adhesion GPCR family, characterized by an extracellular domain containing N-terminal protein modules linked to a seven-transmembrane (7TM) region via a GAIN domain . The antibody targets the extracellular region of human GPR97, specifically residues 98–244AA .

FITC Conjugation

• Mechanism: FITC (fluorescein isothiocyanate) is covalently linked to primary amines (lysines) on the antibody, typically at a ratio of 3–6 FITC molecules per antibody to avoid solubility issues or fluorescence quenching .

• Excitation/Emission: Optimized for 488 nm excitation with emission at 530 nm, suitable for standard fluorescence detection systems .

Role in Neutrophil Activation

GPR97 regulates neutrophil antimicrobial responses via a macromolecular complex involving CD177, mPR3, PAR2, and CD16b . Key findings include:

1. mPR3 Activation: GPR97 allosterically activates membrane-associated protease 3 (mPR3), enhancing its proteolytic activity .

2. PAR2 Signaling: mPR3-mediated cleavage of protease-activated receptor 2 (PAR2) triggers inflammatory responses, including IL-8 upregulation and reactive oxygen species (ROS) production .

3. Antimicrobial Effects: GPR97 ligation increases bacterial uptake and killing in neutrophils .

Signaling Pathways

Optimization Tips

1. Concentration: Use antibody concentrations optimized for flow cytometry (e.g., 1–5 µg/ml) .

2. Background Control: Include isotype-matched controls to assess non-specific binding .

3. Stability: Avoid sodium azide in antibody preparations, as it inhibits FITC conjugation .

Limitations

• Cross-reactivity: Verify species specificity, as some antibodies may bind non-human orthologs .

• Epitope Accessibility: The extracellular targeting region may require membrane permeabilization for intracellular staining .