CY7-SE (Synonyms: Cy7 NHS Ester, N-hydroxysuccinimidyl Ester Cy7)

Labeling Protein^[1]:
Antibodies containing other proteins (e.g., serum albumin or gelatin) or buffers with amino groups can affect labeling and need to be purified before labeling.
1.Dialyze anti-GST antibody (0.5mL at 3mg/mL) in 1L of 0.15M NaCl solution at room temperature for 4 hours.
2.Dialyze overnight in a fresh 1 L of 0.15M NaCl solution at 4°C.
3.On the next day, dialyze for 4 hours in 1L of 0.1M NaHCO₃ (pH 8.3).
4.Filter the antibody solution using a 0.22-μm syringe filter.
5.Dilute a small amount of the antibody with 0.1M NaHCO₃ and measure its UV absorbance at 280nm to calculate the total amount of the antibody.
6.Prepare a Cy7-SE solution in DMSO at a concentration of 10mg/mL. Calculate the required volume to achieve the desired ratio of CyDye NHS to antibody (e.g., 20:1), and then slowly add it to the antibody solution. Stir gently in the dark at room temperature for 45min.
7.Dialyze for 4 hours at room temperature in the dark using 1L of 0.15M NaCl solution to remove unbound Cy7-SE.
8.Dialyze overnight in the dark at 4°C using a fresh 1L of 0.15M NaCl solution.
9.Dialyze for 4 hours at room temperature in the dark using 1L of 0.01M PBS/0.01% sodium azide solution, and then dialyze again overnight at 4°C in the dark.
10.Filter the antibody solution using a 0.22μm syringe filter.
11.Dilute the labeled antibody solution with 0.01M PBS/0.01% sodium azide to a convenient concentration, and measure the UV-Vis absorbance at 280nm (protein) and 552nm (Cy).
12.Dissolve the CY7-SE-labeled protein in PBS solution (pH=7.2-7.4).
13.Intravenous injection of 200μL (0.5mg/mL) into the tail vein of mice.
14.Imaging pictures of in vivo fluorescence were recorded to analyze the distribution of the fluorescent drug.
15.The excitation wavelength was 700–770nm bandpass, and the emission wavelength was 790nm longpass.
To calculate the F/P (fluorophore-to-protein) ratio, follow these steps:
The molar extinction coefficient of Cy7 at 750nm is 240600M⁻¹cm⁻¹; the molar extinction coefficient of this protein at 280nm is 170,000M⁻¹cm⁻¹. Different proteins have different molar extinction coefficients. The absorption of Cy7 dye itself at 280nm is 4% of its absorption at 750nm. The F/P value is calculated using the following formulas：
[Cy7] = A₇₅₀/240600
[antibody] = {A₂₈₀ - (0.04×A₇₅₀)}/170000
F/P _final= [Cy7]/[antibody]= {0.71×A₇₅₀}/ {A₂₈₀ - (0.04×A₇₅₀)}

Reference:
[1] ZHANG C, CHENG H, DOU S, et al. Near-infrared fluorescent molecular probes with cetuximab in the in vivo fluorescence imaging for epithelial ovarian cancer [J]. J Ovarian Res, 2024, 17(1): 225.