AVIL Antibody, FITC conjugated
Description
Introduction
The AVIL Antibody, FITC conjugated, is a specialized immunological reagent designed for detecting Advillin (AVIL), a member of the gelsolin/villin family of actin-regulatory proteins. This article synthesizes data from diverse sources to provide a detailed analysis of its composition, conjugation methodology, applications, and experimental considerations.
AVIL (Advillin) and Its Role
AVIL is a 92 kDa protein expressed in neuronal and epithelial tissues, playing a role in actin remodeling and cellular processes such as neuronal ganglion development . Its structural similarity to villin suggests functional overlap in actin-binding and cytoskeletal regulation .
Key Steps
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Antibody Purification: AVIL antibodies (e.g., rabbit IgG, clone 20956-1-AP) are purified via affinity chromatography to ensure specificity .
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Buffer Optimization: Sodium azide and other interfering agents are removed via dialysis against reaction buffers (e.g., carbonate-bicarbonate, pH 9.5) .
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Fluorochrome Coupling: FITC is conjugated to lysine residues on the antibody via isothiocyanate chemistry. Typical ratios are 40–80 µg FITC/mg antibody, with excess dye removed by gel filtration .
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Quality Control: Fluorescence-to-protein (F/P) ratios are measured spectrophotometrically (A495/A280), with optimal values between 3–6 .
| Parameter | Optimal Range | Impact |
|---|---|---|
| Antibody Concentration | ≥2 mg/mL | Ensures efficient conjugation |
| FITC-to-Antibody Ratio | 40–80 µg/mg | Balances brightness and solubility |
| pH | 9.0–9.5 | Enhances amine reactivity |
Primary Uses
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Immunofluorescence (IF): Detects AVIL in neuronal and epithelial tissues .
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Flow Cytometry: Analyzes AVIL expression in cell populations (e.g., neuronal progenitors) .
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Western Blot (WB): Validates protein expression in lysates (dilution 1:1000–1:4000) .
Secondary Applications
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Biotinylation/avidin systems: FITC-AVIL antibodies can be cross-linked with biotinylated probes for signal amplification .
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Co-localization studies: Combined with other FITC-conjugated markers (e.g., actin) for cytoskeletal analysis .
Challenges and Considerations
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Conjugation Efficiency: Over-labeling (>6 F/P) reduces antibody solubility and specificity .
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Stability: FITC is light-sensitive; conjugates should be stored at 4°C in azide-free buffers .
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Cross-reactivity: Ensure specificity via blocking peptides (e.g., Catalog #AAP32811 for AVIL antibodies) .
Research Findings
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Neuroregeneration: AVIL-FITC antibodies were used to track axonal regeneration in peripheral/central nervous systems .
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Cytoskeletal Dynamics: Co-staining with phalloidin revealed AVIL’s role in actin filament bundling .
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Flow Cytometry Sensitivity: FITC conjugation enhances detection thresholds for rare cell populations (e.g., B1 cells) .
Product Specs
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- NMR structure of the C-terminal headpiece subdomains of advillin. Evaluation of F-actin-binding requirements. PMID: 15096633
- Predicting the effect of a point mutation on a protein fold: the villin and advillin headpieces and their Pro62Ala mutants. PMID: 18022635
