Cy2-SE iodine is a CY dye. CY is the abbreviation of Cyanine, which is a compound composed 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	C29H30IN3O6
Molecular Weight	643.469480037689
Exact Mass	643.117
CAS #	186205-33-4
PubChem CID	121596142
Appearance	Orange to red solid powder
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	8
Rotatable Bond Count	11
Heavy Atom Count	39
Complexity	919
Defined Atom Stereocenter Count	0
SMILES	CCN\1C2=CC=CC=C2O/C1=C\C=C\C3=[N+](C4=CC=CC=C4O3)CCCCCC(=O)ON5C(=O)CCC5=O.[I-]
InChi Key	RQYPAAVMQSMNBY-UHFFFAOYSA-M
InChi Code	InChI=1S/C29H30N3O6.HI/c1-2-30-21-11-5-7-13-23(21)36-27(30)15-10-16-28-31(22-12-6-8-14-24(22)37-28)20-9-3-4-17-29(35)38-32-25(33)18-19-26(32)34;/h5-8,10-16H,2-4,9,17-20H2,1H3;1H/q+1;/p-1
Chemical Name	(2,5-dioxopyrrolidin-1-yl) 6-[2-[(E,3Z)-3-(3-ethyl-1,3-benzoxazol-2-ylidene)prop-1-enyl]-1,3-benzoxazol-3-ium-3-yl]hexanoate;iodide
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: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.
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

Plan 1. Optimal protein preparation: 1) Please prepare the protein (antibody) concentration to 2 mg/mL in order to acquire the labeling effect. 2) The protein solution has a pH of 8.5±0.5. 3) 1M protein should be utilized if the pH is less than 8.0. If the protein concentration is less than 2 mg/mL, the labeling efficiency will be significantly decreased. The range of suggested final protein concentration is 2–10 mg/mL for best labeling efficiency. 4) Birmingham 2 must be transparent for the protein. Preparation of the dye (using CY3-NHS ester as an example): Pour 10 3 of anhydrous DMSO into the CY3-NHS ester vial. The amount of dye (CY3-NHS ester) needed for the reaction is determined by the ideal molar ratio of CY3-NHS to protein as well as the amount of labeled protein. Example: Dissolve 1 mg of CY3-NHS ester in 100 μL of DMSO, assuming that the required labeled protein is 500 μL of 2 mg/mL IgG (MW=150,000). This yields the necessary volume of CY3-NHS ester, which is 5.05 μL. The steps involved in the calculation are 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)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. Execute the coupling reaction 1: Gradually incorporate the recently synthesized 10 mg/mL CY3-NHS ester into the 0.5 mL protein sample solution, give it a gentle shake to combine, quickly centrifuge, and retrieve the sample from the bottom of the reaction tube. 2) For the following sixty minutes, gently flip the reaction tube while keeping it in a dark location. Reverse reaction 5 gradually after ten to fifteen minutes. Conjugates: Using a SepHadex G-25 column, the following methodology demonstrates how to block a dye-conjugate. 1) Assemble the SepHadex G-25 column in accordance with the manufacturer's guidelines. 2) Fill the SepHadex G-25 column with the reaction mixture (from the "Run conjugation reaction"). 3) Add PBS (pH 7.2–7.4) as soon as the sample is running below the top resin surface. 4) To complete column extrusion, add extra PBS (pH 7.2–7.4) to the appropriate sample. Mix the fractions that have the desired conjugate material of dye and protein.

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 : ~62.5 mg/mL (~97.13 mM)

Solubility (In Vivo)

Solubility in Formulation 1: 3.13 mg/mL (4.86 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 31.3 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	1.5541 mL	7.7704 mL	15.5407 mL
	5 mM	0.3108 mL	1.5541 mL	3.1081 mL
	10 mM	0.1554 mL	0.7770 mL	1.5541 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.