Cy7.5 (Cyanine 7.5) is an anthocyanin fluorescent dye that emits near infrared (NIR) fluorescence. Cy7.5 is commonly used for fluorescent protein immunoblotting and live animal imaging.

Protein labeling
I. Solution preparation:
1. Mother solution preparation: Prepare 10 mM Cy7.5 mother solution, store at -20℃ or -80℃ in the dark after aliquoting.
2. Working solution preparation: Select the appropriate working solution concentration according to the experimental requirements, and try to prepare it immediately before use. You can use the condensation solution (500 μg/mL) EDC hydrochloride to activate it before subsequent labeling experiments.
3. Calculation of dye working solution dosage: The amount of CY7.5 dye required for the labeling reaction depends on the amount of protein to be labeled. The optimal molar ratio of CY7.5 dye to protein is about 10;
For example: If the required labeled protein is 500 μL 2 mg/mL IgG (MW=150,000), use 100 μL DMSO to dissolve a tube of 1 mg CY dye, then the required CY volume is 3.95 μL.
2. Protein sample preparation: The recommended protein concentration range is 2-10 mg/mL.
3. Labeling reaction
1. Take a calculated volume of freshly prepared 10 mg/mL CY7.5 dye and slowly add it to 0.5 mL of protein sample solution, gently shake to mix, and then briefly centrifuge to collect the sample at the bottom of the reaction tube. Avoid violent mixing to prevent protein sample denaturation and inactivation;
2. Place the reaction tube in a dark place, gently shake and incubate at room temperature for 60 minutes, and gently invert the reaction tube several times every 10-15 minutes to fully mix the two reactants and improve labeling efficiency.