Cy3 NHS ester  (Synonyms: Cyanine3 NHS ester)

Cy3 NHS ester is a CY dye. CY, short for Cyanine, is a compound consisting of two nitrogen atoms connected by an odd number of methyl units. Cyanine compounds have the characteristics of long wavelength, adjustable absorption and emission, high extinction coefficient, good water solubility and relatively simple synthesis^[1]. CY dyes are of en used for the labeling of proteins, antibodies and small molecular compounds. For the labeling of protein antibodies, the combination can be completed through a simple mixing reaction. Below, we introduce the labeling method of protein antibody labeling, which has certain reference significance^[2].

Guide (The following is our recommended protocol. This protocol is only a guide and should be modified according to your specific needs).
1. Protein Preparation
To obtain the best labeling effect, please prepare the protein (antibody) concentration to 2 mg/mL.
1.1 The pH value of the protein solution should be 8.5 ± 0.5. If the pH is lower than 8.0, adjust it with 1 M sodium bicarbonate.
1.2 If the protein concentration is lower than 2 mg/mL, the labeling efficiency will be greatly reduced. To obtain the best labeling efficiency, it is recommended that the final protein concentration range be 2-10 mg/mL.
1.3 The protein must be in a buffer free of primary amines (such as Tris or glycine) and ammonium ions, otherwise the labeling efficiency will be affected.
2. Dye Preparation
Dilute the dye with anhydrous DMSO to prepare a clear stock solution of 10 mg/mL.
3. Calculation of Dye Amount
The amount of dye required for the reaction depends on the amount of protein to be labeled. The optimal molar ratio of dye to protein is approximately 10:1.
Example: Assuming the required labeled protein is 500 μL 2 mg/mL IgG (MW=150,000), and 1 mg of dye is dissolved in 100 μL DMSO, the required dye volume is 4 μL. The detailed calculation process is as follows:
1) mmol (IgG) = mg/mL (IgG) × mL (IgG)/MW (IgG) = 2 mg/mL × 0.5 mL/150,000 mg/mmol = 6.7 × 10^-6 mmol
2) mmol (dye) = mmol (IgG) × 10 = 6.7 × 10^-6 mmol × 10 = 6.7 × 10^-5 mmol
3) μL (dye) = mmol (dye) × MW (dye)/mg/μL (dye) = 6.7 ×10^-5 mmol ×590.15 mg/mmol/0.01 mg/μL = 4 μL (dye)
Note: We recommend using a dye:protein molar ratio of 10:1. If the concentration is too low or too high, the ratio can be adjusted to 5:1, 15:1, or 20:1.
4. Running the Coupling Reaction
1) Slowly add the calculated volume of freshly prepared 10 mg/mL dye 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 vigorous mixing to prevent denaturation and inactivation of the protein sample.
2) Place the reaction tube in a dark place and incubate gently with shaking for 60 minutes at room temperature. Every 10-15 minutes, gently invert the reaction tube several times to thoroughly mix the reactants and improve labeling efficiency.
5. Purification of Conjugates
The following protocol is an example of purifying dye-protein conjugates using a Labelling Kits Centrifugation-Based Rapid Desalting Column (5KD) (HY-D3014).
5.1 Prepare the desalting column according to the instructions.
5.2 Load the reaction mixture onto the top of the desalting column.
5.3 Once the sample has run below the resin surface, immediately add PBS (pH 7.2-7.4).
5.4 Add more PBS (pH 7.2-7.4) to the target sample to complete column purification. Collect the fraction containing the desired dye-protein conjugate.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

590.15

C₃₄H₄₀ClN₃O₄

1393363-07-9

Solid

Brown to breen

570

550

CC1(C)C(/C=C/C=C2N(C)C3=C(C=CC=C3)C/2(C)C)=[N+](CCCCCC(ON4C(CCC4=O)=O)=O)C5=C1C=CC=C5.[Cl-]

Room temperature in continental US; may vary elsewhere.

-20°C, protect from light, stored under nitrogen

*In solvent : -80°C, 6 months; -20°C, 1 month (protect from light, stored under nitrogen)

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (protect from light, stored under nitrogen). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

• [1]. Ptaszek M. Rational design of fluorophores for in vivo applications. Prog Mol Biol Transl Sci. 2013;113:59-108.  [Content Brief]

  [2]. Shindy, H. A. (2017). Fundamentals in the chemistry of cyanine dyes: A review. Dyes and Pigments, 145, 505–513. doi:10.1016/j.dyepig.2017.06.029