Cyanine3.5 maleimide chloride is a type of CY dye, where CY stands for cyanine, a compound with two nitrogen atoms linked by an odd number of methine units. Cyanine compounds are known for their long wavelengths, adjustable absorption and emission, high extinction coefficients, good water solubility, and relatively straightforward synthesis. CY series dyes are frequently used for labeling proteins, antibodies, and small molecules. Protein and antibody labeling can be achieved through a simple mixing reaction. Below, we outline a method for labeling proteins and antibodies, which may serve as a useful reference.

Preparation of Stock Solution: 1. Protein Preparation: To achieve optimal labeling, prepare protein (antibody) at a concentration of 2 mg/mL. 1) Ensure the protein solution's pH is 8.5±0.5; if below 8.0, adjust with 1 M sodium bicarbonate. 2) If protein concentration is below 2 mg/mL, labeling efficiency drops significantly; optimal protein concentration is recommended to be between 2-10 mg/mL. 2. Dye Preparation: Dilute CY dye with anhydrous DMSO to create a 10 mM stock solution, mixing thoroughly via glass pipette or vortex. Note: Store CY stock solution in aliquots at -20 ℃ or -80 ℃, away from light. 3. Calculation of Dye Working Solution: The amount of CY dye required for labeling depends on protein amount; the optimal molar ratio of CY dye to protein is approximately 10. Example: For 500 μL of 2 mg/mL IgG (MW=150,000), dissolve 1 mg CY dye in 100 μL DMSO, requiring 3.95 μL CY dye. Calculations (using CY3-NHS ester as an example): 1) mmol (IgG) = 2 mg/mL × 0.5 mL / 150,000 mg/mmol = 6.7×10⁻⁶ mmol 2) mmol (CY3-NHS ester) = 6.7×10⁻⁶ mmol × 10 = 6.7×10⁻⁵ mmol 3) μL (CY3-NHS ester) = 6.7×10⁻⁵ mmol × 590.15 mg/mmol / 0.01 mg/μL = 3.95 μL (CY3-NHS ester). Usage Method: 1. Labeling Reaction: 1) Slowly add freshly prepared CY dye (10 mg/mL) to 0.5 mL protein solution, mix gently, then briefly centrifuge to collect the sample at the tube's bottom; avoid vigorous mixing to prevent protein denaturation. 2) Incubate the reaction tube in the dark at room temperature, gently shaking for 60 minutes, inverting the tube every 10–15 minutes to mix reagents and enhance labeling efficiency. 2. Protein Purification and Desalting: Use a SepHadex G-25 column to purify dye-protein conjugates. 1) Prepare SepHadex G-25 column as per the manufacturer's instructions. 2) Load the reaction mixture onto the column. 3) When the sample reaches the top of the resin, immediately add PBS (pH 7.2-7.4). 4) Add more PBS (pH 7.2-7.4) to complete column purification, collecting fractions containing desired dye-protein conjugates. Optional: If the protein lacks free cysteines, use reducing agents like DTT or TCEP to reduce disulfide bonds to free thiol groups. When using DTT, remove free DTT through dialysis before conjugating maleimide dyes with the protein.