cyn-5 Antibody
Description
Definition and Structure of Cy5 Antibodies
Cy5 antibodies fall into two categories:
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Cy5-conjugated antibodies: Antibodies chemically linked to Cy5 fluorophores for direct target detection ( ).
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Anti-Cy5 antibodies: Antibodies that specifically bind to Cy5 tags, enabling signal amplification in assays (e.g., ).
Cy5 is a synthetic cyanine dye with excitation/emission maxima at 633/680 nm, making it compatible with red laser-equipped instruments like flow cytometers ( ). Its small molecular size (~1 kDa) allows conjugation without significantly altering antibody function.
Conjugation Protocols and Optimization
Cy5 is typically conjugated to antibodies via primary amines (lysine residues) under controlled conditions:
Key Conjugation Parameters
| Parameter | Optimal Value | Purpose |
|---|---|---|
| Antibody concentration | ≥2 mg/ml | Ensures efficient dye-to-antibody ratio |
| Molar ratio (Cy5:IgG) | 5:1 | Balances brightness and solubility |
| Reaction time | 1 hour (RT, dark) | Maximizes covalent bond formation |
| Buffer | Amine-free (pH 6.5–8.5) | Prevents competing reactions |
Post-conjugation, unbound Cy5 is removed via gel filtration or dialysis. Suboptimal ratios (>7:1) risk quenching fluorescence or antibody aggregation ( ).
Apoptosis Detection
Cy5 Annexin V binds phosphatidylserine (PS) exposed on apoptotic cell membranes. When paired with 7-AAD (a viability dye), it distinguishes:
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Viable cells: Cy5⁻/7-AAD⁻
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Early apoptotic cells: Cy5⁺/7-AAD⁻
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Late apoptotic/dead cells: Cy5⁺/7-AAD⁺
Example protocol (Jurkat cells):
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Induce apoptosis with 4–6 µM camptothecin for 4–6 hours.
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Stain with Cy5 Annexin V and 7-AAD.
Signal Amplification
Anti-Cy5 monoclonal antibodies (e.g., clones B-2 or CY5-15 ) enhance detection sensitivity in:
Critical Considerations
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Sodium azide interference: Must be removed from antibodies pre-conjugation to avoid dye inactivation ( ).
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Adherent cell limitations: Mechanical harvesting can artifactually increase PS exposure, necessitating gentle detachment methods ( ).
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Storage: Cy5 conjugates degrade under repeated freeze-thaw cycles; aliquot in glycerol-containing buffers ( ).
Research Findings and Validation
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Cy5-conjugated antibodies exhibit ~95% recovery post-purification when using optimized conjugation kits (e.g., LYNX Rapid RPE-Cy5.5 Kit ).
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In apoptosis assays, Cy5 Annexin V detects PS exposure 4–6 hours post-treatment, preceding membrane rupture ( ).
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Anti-Cy5 antibodies enable multiplexed imaging by recognizing synthetic Cy5 tags introduced via secondary probes ( ).
Troubleshooting Guide
| Issue | Solution |
|---|---|
| High background fluorescence | Reduce antibody dilution (1:1,000–1:5,000) |
| Low conjugation efficiency | Use fresh DMSO for Cy5 dissolution |
| Cross-reactivity | Pre-adsorb with Cy5-free serum |
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- The most potent derivative exhibits a binding affinity (Kd) of 11.2 ± 9.2 µM to cyclophilin A (CypA) and an IC50 value of 190 µM for activity against Caenorhabditis elegans (C. elegans). This compares to an IC50 of 28 µM for cyclophilin A. PMID: 17927958
