CY2-SE is a fluorescent labeling reagent that targets primary amine groups (-NH2) present in proteins, peptides, and oligonucleotides. The N-hydroxysuccinimidyl (NHS) ester moiety reacts with nucleophilic primary amines under mild alkaline conditions (pH 7-9) to form stable, covalent amide bonds. This reaction mechanism enables the covalent attachment of the Cy2 fluorophore to target biomolecules, facilitating their detection, tracking, and quantification in various experimental systems.

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.

---

CY2-SE exhibits excitation and emission maxima at 473 nm and 510 nm, respectively. The compound is supplied as a crystalline solid with a purity of ≥98% and is soluble in DMF, DMSO, and DMF:PBS mixtures. As an amine-reactive probe, CY2-SE efficiently labels amino groups on target biomolecules through NHS ester chemistry, forming stable covalent bonds. The optimal molar ratio of CY2-SE dye to protein for labeling is approximately 10:1.

CY2-SE is not typically used for in vivo applications due to its reactive NHS ester chemistry which would label multiple proteins non-specifically. The compound is primarily used for in vitro labeling of purified proteins, antibodies, and other biomolecules for downstream applications including fluorescence microscopy, flow cytometry, Western blotting, and other detection methods.

Protein labeling with CY2-SE: Prepare the protein (antibody) at a concentration of 2 mg/mL in buffer at pH 8.5+/-0.5. Dissolve CY2-SE in anhydrous DMF or DMSO at an appropriate concentration. Add the dye solution to the protein solution at a molar ratio of approximately 10:1 (dye:protein). Incubate the reaction mixture at room temperature for 1-2 hours with gentle mixing. Remove unreacted dye by dialysis, gel filtration, or size exclusion chromatography. Determine the labeling efficiency by measuring absorbance at 280 nm (protein) and 486 nm (Cy2).

CY2-SE is used for labeling proteins and antibodies for in vitro applications. Cells or tissues are incubated with CY2-SE-labeled probes for detection by fluorescence microscopy or flow cytometry. For antibody labeling, purified antibodies are incubated with CY2-SE under the same conditions as described for protein labeling. Labeled antibodies are then used for immunofluorescence staining of fixed cells or tissue sections. For oligonucleotide labeling, amine-modified oligonucleotides are reacted with CY2-SE in appropriate buffers.

CY2-SE is not used for in vivo animal studies due to its reactive nature. The compound is strictly an in vitro labeling reagent. For animal studies, pre-labeled biomolecules (e.g., CY2-SE-labeled antibodies or peptides) may be administered to track distribution or target engagement, but the dye itself is not administered directly to animals. The labeled probes are typically administered via intravenous or intraperitoneal injection at doses determined by the specific experimental requirements.

CY2-SE is not a therapeutic compound and pharmacokinetic data are not applicable. The compound is a research reagent used for labeling biomolecules in vitro. Once conjugated to a protein or antibody, the pharmacokinetics of the labeled probe would be determined by the carrier molecule rather than the dye itself. CY2-SE is not intended for in vivo administration as a free compound.

CY2-SE is an amine-reactive fluorescent probe and is not intended for therapeutic use. Toxicity data for the compound itself are limited as it is used as a labeling reagent rather than a therapeutic agent. Standard laboratory safety practices should be followed when handling CY2-SE, including the use of appropriate personal protective equipment. The compound should be handled in a fume hood, and contact with skin and eyes should be avoided. Disposal should follow institutional guidelines for chemical waste.

[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.

CY2-SE (Cyanine2 Succinimidyl Ester) is a fluorescent labeling reagent used for the covalent modification of primary amines on proteins, peptides, and oligonucleotides. It exhibits excitation/emission maxima of 473/510 nm. The NHS ester functionality enables efficient labeling under mild conditions. CY2-SE is widely used in biochemical research for fluorescence-based detection and imaging applications. The compound is for research use only and is not approved for diagnostic or therapeutic applications.

C29H30IN3O6

643.469480037689

643.117

186205-33-4

121596142

Orange to red solid powder

0

8

11

39

919

0

CCN\1C2=CC=CC=C2O/C1=C\C=C\C3=[N+](C4=CC=CC=C4O3)CCCCCC(=O)ON5C(=O)CCC5=O.[I-]

RQYPAAVMQSMNBY-UHFFFAOYSA-M

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

(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

2934.99.9001

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.

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

DMSO : ~62.5 mg/mL (~97.13 mM)

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.)