Sulfo-Cy5.5 is a near-infrared fluorescent dye (Ex=673 nm, Em=707 nm) that can be used to label biomolecules such as peptides, proteins, and oligonucleotides [2].

Prepare the stock solution
1. Protein Preparation
For optimal labeling, the protein (antibody) concentration needs to be adjusted to 2 mg/mL.
1) Ensure that the pH of the protein solution is within the range of 8.5±0.5. If the pH is below 8.0, adjust with 1 M sodium bicarbonate.
2) Labeling efficiency is significantly reduced when the protein concentration is below 2 mg/mL. To optimize the labeling effect, it is recommended to maintain the protein concentration between 2-10 mg/mL.
3) Protein should be dissolved in buffer without primary amines (e.g. Tris or glycine) and ammonium ions, otherwise it will interfere with the labeling reaction.
2. Dye preparation
Prepare Sulfo-Cy5.5 dye using anhydrous DMSO in a 10 mM stock solution. Mix by glass rod or vortexing.
Note: The Sulfo-Cy5.5 dye stock solution is recommended to be dispensed and stored at -20 °C or -80 °C, protected from light. Before subsequent labeling experiments, activation with 500 μg/mL of Condensation Solution (EDC hydrochloride T19947) is required.
3. Calculation of the amount of dye working solution
The amount of Sulfo-Cy5.5 dye to be used depends on the amount of protein to be labeled; the optimal molar ratio of Sulfo-Cy5.5 dye to protein is approximately 10.
The optimal molar ratio of CY dye to protein is about 10. Example: 500 μL of 2 mg/mL IgG (MW = 150,000) is to be labeled. Assuming that 1 mg of CY dye is dissolved in 100 μL of DMSO, the calculation is as follows:
1) IgG (mmol) = IgG (mg/mL) × IgG (mL) / IgG (MW) =2 mg/mL×0.5 mL / 150000 mg/mmol= 6.7×10^6 mmol
2) Sulfo-Cy5.5 (mmol) = IgG (mmol) ×10 =6.7×10^6 mmol×10 = 6.7×10^5 mmol
3) Sulfo-Cy5.5 (μL) = Sulfo-Cy5.5 (mmol) ×Sulfo-Cy5.5 (MW) / mg/Sulfo-Cy5.5 (μL) = 6.7×10^5 mmol× 917.05 g/mol / 0.01 mg/μL
Labeling reaction
1) Take a calculated amount of freshly prepared 10 mM Sulfo-Cy5.5 dye master mix (approx. 10 μL) and add 50 μL of 500 μg/mL condensation solution for activation. Slowly add this mixture to 0.5 mL of protein sample solution, mix gently and centrifuge briefly to allow the sample to settle to the bottom of the reaction tube. Avoid violent shaking to avoid protein denaturation or inactivation.
2) Place the reaction tube in a light-proof environment and incubate for 60 minutes at room temperature with gentle shaking. Gently turn the tube over several times every 10-15 minutes to ensure adequate mixing and improve labeling efficiency.