RFNG Antibody, FITC conjugated

Definition and Purpose

RFP Antibody, FITC Conjugated is a polyclonal antibody raised against Red Fluorescent Protein (RFP) derived from Discosoma species, chemically linked to FITC for fluorescence-based detection . FITC, a small organic fluorophore, emits green light (~525 nm) when excited at 495 nm, enabling visualization of target proteins in assays like immunofluorescence (IF), Western blotting (WB), and flow cytometry .

Key characteristics:

• Target: RFP and its variants (e.g., DsRed, drFP583) .

• Host: Rabbit .

• Conjugate: FITC (fluorescein isothiocyanate) .

• Applications: Immunofluorescence, WB, ELISA, and fluorescence-activated cell sorting (FACS) .

Immunofluorescence (IF) and Microscopy

• Detects RFP-tagged proteins in fixed/permeabilized cells with high signal-to-noise ratio .

• Enables co-localization studies when paired with other fluorophores (e.g., DyLight 633) .

Western Blotting

• Amplifies signal via secondary antibody binding (1:500 dilution recommended) .

Flow Cytometry

• Validated for FACS analysis, with minimal background staining in control experiments .

Performance and Validation

• Specificity: Binds RFP variants with minimal cross-reactivity .

• Sensitivity: Detects RFP at concentrations as low as 1 µg/mL in ELISA .

• Linearity: Fluorescence intensity correlates linearly with target protein concentration within dynamic range .

Comparative Advantages Over Alternative Probes

Research Findings

• Conjugation Heterogeneity: Lysine-based FITC labeling produces a mixture of conjugates (2–6 FITC molecules per antibody), impacting signal intensity .

• Quenching Mitigation: Optimal FITC-to-antibody ratios (40–80 µg FITC per mg antibody) reduce internal quenching .

• Validation: Co-localization studies with anti-integrin β3 antibodies confirm specificity in tumor tissues .

Limitations and Considerations

• Sodium Azide Interference: Residual azide in antibody preparations inhibits FITC conjugation; thorough dialysis is required pre-labeling .

• Photobleaching: FITC fluorescence degrades under prolonged light exposure .

• Batch Variability: Polyclonal nature may lead to slight inter-batch differences .