Cy3 NHS ester is a CY dye. CY is the abbreviation of Cyanine, which 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. CY dyes are often used for labeling proteins, antibodies and small molecule compounds. For labeling protein antibodies, the binding can be completed through a simple mixing reaction. Below we introduce the labeling method for protein antibody labeling, which has certain reference significance. .

Physicochemical Properties

Molecular Formula	C34H40CLN3O4
Molecular Weight	590.1521
Exact Mass	589.27
CAS #	1393363-07-9
PubChem CID	155487015
Appearance	Light yellow to green yellow solid powder
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	10
Heavy Atom Count	42
Complexity	1120
Defined Atom Stereocenter Count	0
SMILES	[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
InChi Key	KWDMHANYPGHTLW-UHFFFAOYSA-M
InChi Code	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
Chemical Name	(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
HS Tariff Code	2934.99.9001
Storage	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.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

ln Vitro

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.

References

[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.

Solubility Data

Solubility (In Vitro)

DMSO: 125 mg/mL (211.81 mM)

Solubility (In Vivo)

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

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.6945 mL	8.4724 mL	16.9448 mL
	5 mM	0.3389 mL	1.6945 mL	3.3890 mL
	10 mM	0.1694 mL	0.8472 mL	1.6945 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.