Cy5-bifunctional dye

Cy5-bifunctional dye is a bifunctional dye used for covalent labeling of primary amines on proteins or oligonucleotides (Ex/Em = 649 nm/670 nm). Cy5-bifunctional dye can label recombinant annexin-V to assess phosphatidylserine exposure on the cell surface via flow cytometry. Cy5-bifunctional dye can label anti-human IgG (H + L) secondary antibodies. Cy5-bifunctional dye is applicable to studies of glanders and melioidosis^[1]. In Vitro:Guide (The following is our recommended solution. This solution is merely a guideline and should be modified according to your specific needs.)
1.Protein Preparation: Ensure the protein is in an amine-free buffer (e.g., PBS or bicarbonate buffer) at pH 8.5-9.0, typically at a concentration of 2-10 mg/mL.
2.Dye Preparation: Dissolve the Cy5-bifunctional dye in anhydrous DMSO to a concentration of 10 mg/mL immediately before use.
3.Reaction: Add the Cy5 dye solution to the protein solution while stirring. A common molar ratio is 12:1 (Cy5:Protein), but this should be optimized based on the desired degree of labeling.
4.Incubation: Incubate the mixture at room temperature for 60 minutes in the dark.
5.Purification: Separate the labeled protein from unreacted dye using gel filtration chromatography (e.g., Sephadex G-25).
Considerations:
(1)\tOptimal pH: The reaction is pH-sensitive; primary amines must be unprotonated, making pH 8.5–9.0 necessary.
(2)\tQuenching: Cy5 can show significant quenching at high labeling ratios, which is more pronounced than with Cy3.
(3)\tStability: Store Cy5-bifunctional dye stock solutions in DMSO at -20°C, protected from light.
(4)\tPurification: Proper removal of unreacted dye is critical for accurate fluorescence measurement, often validated by calculating the absorbance ratio (A₇₅₅:A₅₆₅ for PE-Cy5).