The NHS ester functional group on Cy3 NHS ester targets primary amines (-NH₂) on proteins, antibodies, peptides, and nucleic acids. The compound does not target a specific protein or enzyme but rather serves as a covalent labeling reagent that attaches the Cy3 fluorophore to amine-containing biomolecules. Cy3 is a cyanine dye consisting of two nitrogen atoms connected by an odd number of methyl units. Cyanine compounds have long wavelength, adjustable absorption and emission, high extinction coefficient, good water solubility, and relatively simple synthesis.

First Protocol. Protein preparation: Please prepare the protein (antibody) concentration to 2 mg/mL in order to achieve the greatest labeling effect. 2) The protein solution has a pH of 8.5±0.5. One milligram of sodium bicarbonate (1M) should be added to the pH if it is less than 8.0. 3) The labeling efficiency will be significantly decreased if the protein content is less than 2 mg/mL. The range of suggested final protein concentration is 2–10 mg/mL for best labeling efficiency. 4) To ensure optimal labeling efficacy, the protein needs to be in a buffer free of ammonium ions and primary amines, like Tris or glycine. 2. Prepare the dye (using CY3-NHS ester as an example). Fill the vial containing the CY3-NHS ester with anhydrous DMSO to create a 10 mM stock solution. Use a pipette or vortex to thoroughly mix. 3. dye dose computation. The ideal molar ratio of CY3-NHS ester to protein is approximately 10, and the amount of CY3-NHS ester needed for the reaction depends on the amount of protein to be labeled. Example: Dissolve 1 mg of CY3-NHS ester in 100 μL of DMSO, assuming that the required marker protein is 500 μL 2 mg/mL IgG (MW=150,000). This yields the 5.05 μL of CY3-NHS ester that is needed. The exact calculation procedure 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 (CY3-NHS ester) = mmol (IgG) × 10=6.7×10-6 mmol×10=6.7 × 10-5 mmol 3) uL (CY3-NHS ester) = mmol (CY3-NHS ester) ×MW (CY3-NHS ester)/mg/μL (CY3-NHS ester) = 6.7 ×10-5 mmol ×753.88 mg/mmol/0.01 mg/μL=5.05 μL (CY3-NHS ester) 4. 1) Perform the coupling reaction: Take a 0.5 mL protein sample in the solution, add an appropriate amount of freshly made 10 mg/mL CY3 -NHS ester, shake gently to mix, and then centrifuge for a brief period of time to collect the sample at the tub's bottom. To prevent denaturation and inactivation of protein samples, do not mix them equally. 2) After placing the reaction tube in a dark location, gently incubate it for 60 minutes at room temperature. After ten to fifteen minutes, carefully reverse step five. Purify the conjugate: Using a SepHadex G-25 column, the following process demonstrates how to purify a dye-protein conjugate. 1) Assemble the SepHadex G-25 column in accordance with the manufacturer's guidelines. 2) Fill the SepHadex G-25 column to the brim with the reaction mixture (from "Run conjugation reaction"). 3) Add PBS (pH 7.2–7.4) as soon as the sample is running below the top resin surface. To finish the purification of the chosen sample, add additional PBS (pH 7.2–7.4). The appropriate dye-protein conjugate-containing fractions were combined.

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The in vitro activity of Cy3 NHS ester is its function as a fluorescent labeling reagent. The NHS ester reacts with primary amines to form stable amide bonds. The Cy3 fluorophore provides bright orange-red fluorescence, making labeled conjugates detectable by fluorescence microscopy, flow cytometry, and FRET studies. Cy3 is often used as a donor in FRET applications. The labeling is efficient and can be completed through a simple mixing reaction.

Cy3 NHS ester is not used as a therapeutic agent. Its in vivo utility is limited to research applications involving the fluorescent labeling of biomolecules for imaging and detection. The compound is not intended for systemic therapeutic use.

In vitro labeling assays for Cy3 NHS ester involve incubating the NHS ester with amine-containing biomolecules in appropriate buffer conditions (typically pH 7-9). The reaction proceeds through nucleophilic attack of the primary amine on the NHS ester carbonyl, forming a stable amide bond and releasing N-hydroxysuccinimide as a byproduct. Labeling efficiency can be assessed by measuring the fluorescence intensity of the conjugated product using fluorescence spectroscopy or by determining the dye-to-protein ratio.

For in vitro cellular experiments, Cy3 NHS ester is first conjugated to antibodies, proteins, or other targeting ligands via the NHS ester-amine reaction. The labeled conjugates are then purified and added to cell culture medium for incubation with target cells. After washing, cells are analyzed by fluorescence microscopy or flow cytometry. The orange-red fluorescence provides bright and photostable labeling for cellular imaging.

In vivo animal experiments with Cy3 NHS ester are not typically performed with the compound itself. However, conjugates prepared with this dye can be used for imaging applications in animal models.

Pharmacokinetic properties of Cy3 NHS ester have not been characterized, as the compound is a research-use fluorescent labeling reagent rather than a drug candidate. The compound has a molecular weight of 590.15 g/mol and should be stored at -20°C in the dark.

Cy3 NHS ester is intended for research use only and not for human therapeutic or diagnostic applications. Standard laboratory safety precautions should be observed when handling this chemical reagent.

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

[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.

Cy3 NHS ester (CAS#: 1393363-07-9) is an amine-reactive orange-red fluorescent dye used for labeling proteins, antibodies, and nucleic acids. It is used in fluorescence microscopy, flow cytometry, and FRET studies. The compound has no clinical or therapeutic applications.

C34H40CLN3O4

590.1521

589.27

1393363-07-9

155487015

Light yellow to green yellow solid powder

0

6

10

42

1120

0

[Cl-].O(C(CCCCCN1C2=CC=CC=C2C(C)(C)/C/1=C/C=C/C1C(C)(C)C2=CC=CC=C2[N+]=1C)=O)N1C(CCC1=O)=O

KWDMHANYPGHTLW-UHFFFAOYSA-M

InChI=1S/C34H40N3O4.ClH/c1-33(2)24-14-8-10-16-26(24)35(5)28(33)18-13-19-29-34(3,4)25-15-9-11-17-27(25)36(29)23-12-6-7-20-32(40)41-37-30(38)21-22-31(37)39;/h8-11,13-19H,6-7,12,20-23H2,1-5H3;1H/q+1;/p-1

(2,5-dioxopyrrolidin-1-yl) 6-[(2Z)-3,3-dimethyl-2-[(E)-3-(1,3,3-trimethylindol-1-ium-2-yl)prop-2-enylidene]indol-1-yl]hexanoate;chloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 125 mg/mL (211.81 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.52 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.52 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)