Cy5.5-SE is a CY5.5-derived dye. CY5.5 is an amine-reactive, near-infrared (NIR) fluorophore with Ex/Em = 675/695 nm, frequently employed for labelling nucleic acids and proteins.

Reference for Using Cy5.5-SE:
1. Protein Preparation
It is recommended to prepare the protein (antibody) at a concentration of 2 mg/mL, with the pH adjusted to 8.5 ± 0.5. If the pH drops below 8.0, adjust it using 1 M sodium bicarbonate.
Note: If the protein concentration is below 2 mg/mL, labeling efficiency will decrease significantly. For optimal labeling, keep the protein concentration between 2–10 mg/mL. The protein buffer should not contain primary amines (like Tris or glycine) or ammonium ions, as these interfere with labeling.
2. Dye Preparation
Dissolve Cy5.5-SE dye in anhydrous DMSO to make a 10 mg/mL stock solution. Aliquot and store the solution at -20°C or -80°C, protected from light.
3. Calculating Dye Amount
The amount of Cy5.5-SE dye needed depends on the amount of protein to be labeled. The ideal molar ratio of Cy5.5-SE dye to protein is approximately 10.
Example: If you’re labeling 500 μL of 2 mg/mL IgG (MW = 150,000), and 1 mg of Cy5.5-SE dye is dissolved in 100 μL DMSO, then the required volume of Cy5.5-SE dye is 6.79 μL. The detailed calculation is:
1) mmol (IgG) = mg/mL (IgG) × mL (IgG) / MW (IgG) = 2 mg/mL × 0.5 mL / 150,000 mg/mmol = 6.7 × 10⁻⁶ mmol
2) mmol (Cy5.5-SE) = mmol (IgG) × 10 = 6.7 × 10⁻⁶ mmol × 10 = 6.7 × 10⁻⁵ mmol
3) μL (Cy5.5-SE) = mmol (Cy5.5-SE) × MW (Cy5.5-SE) / mg/μL (Cy5.5-SE) = 6.7 × 10⁻⁵ mmol × 1014.13 mg/mmol / 0.01 mg/μL = 6.79 μL
4. Labeling Reaction
Gradually add the calculated volume of 10 mg/mL CY dye to 0.5 mL of the protein solution. Mix gently and briefly centrifuge to collect the sample at the bottom of the tube. Incubate in the dark at room temperature with gentle shaking for 60 minutes. Invert the tube several times every 10–15 minutes.
5. Protein Purification
Purify the dye-protein conjugate using an appropriate method.

Cy5.5-labeled factor VIIa was developed for tumor imaging: These targeted proteins can specifically accumulate in tumor parazacco spilurus subsp. spilurus grafts for at least 14 days, whereas free Cy5.5 does not accumulate in any grafts or organs. This tumor VEC anti-tissue factor-based imaging strategy enables detection of primary and metastatic lesions and monitoring of therapeutic responses in vivo [1]. Additionally, pH/temperature-sensitive magnetic nanogels conjugated with Cy5.5-labeled lactoferrin (Cy5.5-Lf-MPNA nanogels) were designed as promising contrast agents for preoperative MRI and intraoperative fluorescence imaging of glioma [2].