CY5-SE (Cy5 NHS Ester) is a reactive dye used to label amino groups in peptides, proteins and nucleotides. It needs to be stored away from light. Excitation wavelength (nm): 640, emission wavelength (nm): 664.

1 mL of each 100 μg/mL SELP solution in 100 mM sodium bicarbonate buffer (pH 8.3) was mixed with 10 μL of 1.2 mg/mL Cy5 mono NHS-ester（CY5-SE） in 10% dimethyl sulfoxide and incubated for 2 h on ice. To quench the reaction, 50 μL of 1 M Tris-HCl (pH 8.0) was added to the reaction solution. To calculate molar concentrations of Cy5 and SELP in the Cy5-conjugated SELPs, reactions containing designated concentrations of pure SELP and BCA solution were conducted for 30 min at 37 °C. The absorbances of SELP and Cy5 at 562 and 633 nm were then obtained using the Microplate reader. [1]

I. Preparation of stock solution
1. Protein preparation
1) To achieve the best labeling effect, adjust the protein (antibody) concentration to 2 mg/mL.
2) Ensure that the pH of the protein solution is within the range of 8.5±0.5. If the pH value is lower than 8.0, 1 M sodium bicarbonate can be used to adjust.
3) When the protein concentration is lower than 2 mg/mL, the labeling efficiency will be significantly reduced. To optimize the labeling effect, it is recommended to maintain the protein concentration between 2-10 mg/mL.
4) The protein needs to be dissolved in a buffer that does not contain primary amines (such as Tris or glycine) and ammonium ions, otherwise it will interfere with the labeling reaction.
2. Dye preparation
Use anhydrous DMSO to prepare a 10 mM stock solution of CY5-SE dye. Mix well with a glass rod or vortex.
Note: It is recommended that the CY5-SE dye stock solution be stored at -20 ℃ or -80 ℃ in the dark after aliquoting. Before the subsequent labeling experiment, it is necessary to activate it with 500 μg/mL condensation solution (EDC hydrochloride T19947).
3. Calculation of the amount of dye working solution
The amount of CY5-SE dye used depends on the amount of labeled protein. The optimal molar ratio of CY5-SE dye to protein is about 10.
Example: 500 μL, 2 mg/mL IgG (MW = 150,000) needs to be labeled. Assuming that 100 μL DMSO is used to dissolve 1 mg of CY dye, 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) CY5-SE (mmol) = IgG (mmol) ×10 =6.7×10^6 mmol×10 = 6.7×10^5 mmol
3) CY5-SE (μL) = CY5-SE(mmol)×CY5-SE(MW)/mg/CY5-SE (μL) = 6.7×10^5 mmol× 753.88.06 g/mol / 0.01 mg/μL
II. Labeling reaction
1) Take the calculated amount of freshly prepared 10 mM CY5-SE dye stock solution (approximately 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, gently mix and briefly centrifuge to allow the sample to sink to the bottom of the reaction tube. Avoid vigorous shaking to prevent protein denaturation or inactivation.
2) Place the reaction tube in a dark environment and incubate at room temperature with gentle shaking for 60 minutes. Gently flip the reaction tube several times every 10-15 minutes to ensure adequate mixing and improve labeling efficiency.