Cy7.5 maleimide chloride is a type of CY dye, where CY stands for the cyanine class of compounds, consisting of 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 simple synthesis. CY dyes are frequently used to label proteins, antibodies, and small molecular compounds. For antibody labeling, the dyes can be conjugated through a straightforward mixing reaction, making them a useful reference for protein and antibody labeling methods.

To prepare the original solution, start with the preparation of protein. For optimal labeling, the protein (antibody) concentration should be set to 2 mg/mL. Ensure the protein solution has a pH of 8.5±0.5, and adjust with 1 M sodium bicarbonate if it falls below pH 8.0. Protein concentration lower than 2 mg/mL significantly reduces labeling efficiency, so it is recommended to maintain a final concentration between 2-10 mg/mL. The protein must be in a buffer free of primary amines (e.g., Tris or glycine) and ammonium ions to avoid affecting the labeling process. For dye preparation, dilute the CY dye in anhydrous DMSO to create a 10 mM stock solution, mixing thoroughly via a glass pipette or vortex. Note: CY stock should be aliquoted and stored in the dark at -20 ℃ or -80 ℃. Calculate the working dye solution based on the protein amount to be labeled, maintaining an optimal molar ratio of 10 between the CY dye and the protein. For example, to label 500 μL of 2 mg/mL IgG (MW=150,000), dissolve 1 mg of CY dye in 100 μL DMSO, requiring 3.95 μL of CY. Calculation steps for CY3-NHS ester: 1) mmol (IgG) = (mg/mL × mL) / MW = 2 mg/mL × 0.5 mL / 150,000 mg/mmol = 6.7×10⁻⁶ mmol; 2) mmol (CY3-NHS ester) = mmol (IgG) × 10 = 6.7×10⁻⁶ mmol × 10 = 6.7×10⁻⁵ mmol; 3) μL (CY3-NHS ester) = mmol × MW / mg/μL = 6.7×10⁻⁵ mmol × 590.15 mg/mmol / 0.01 mg/μL = 3.95 μL. For usage, add the calculated fresh 10 mg/mL CY dye volume slowly to 0.5 mL of protein solution, mix gently, and briefly centrifuge. Avoid vigorous mixing to prevent protein denaturation. Incubate the tube in the dark at room temperature for 60 minutes, gently inverting every 10–15 minutes to enhance reaction mixing and labeling efficiency. For protein purification and desalting, use a Sephadex G-25 column: 1) Prepare according to manufacturer's instructions. 2) Load the reaction mixture onto the column. 3) Add PBS (pH 7.2-7.4) when the sample reaches just below the resin surface, proceed with more PBS to finish column purification, and collect the desired dye-protein conjugate fractions.