The target of Cy5 maleimide is thiol groups (-SH) in proteins and peptides, particularly cysteine residues. Its mechanism of action is based on the Michael addition reaction between the maleimide group and the thiolate anion of cysteine under mild conditions (pH 6.5-7.5). This reaction forms a stable thioether bond, covalently attaching the Cy5 fluorophore to the target protein. The Cy5 dye provides bright red/near-infrared fluorescence, which is advantageous for imaging applications due to reduced background autofluorescence in biological samples.

The optimal way to prepare stock solution 1. Protein preparation: To get the desired labeling effect, add 2 mg/mL of protein (antibody). The ideal pH for the protein solution is 8.5±0.5. Use 1 M carbon dioxide if the pH is less than 8.0. 2) A protein concentration below 2 mg/mL will result in significantly poorer labeling efficiency. Finally, it is advised to use a protein concentration range of 2–10 mg/mL in order to achieve the best labeling efficiency. 3) To ensure maximum labeling efficiency, the protein needs to be in a transparent buffer that contains ammonium ions and primary amines, like Tris or glycine. 2. Dye preparation: Make a 10 mM stock solution of CY dye by adding anhydrous DMSO. Blend well using a glass tube or a vortex. After being aliquoted, CY storage solution should be kept in the dark at -20°C or -80°C. 3. Amount of working solution for dye The amount of CY dye needed for the labeling reaction is determined by the amount of protein that has been marked. The ideal ratio for CY dye and protein is as follows: let's say 500 μL of 2 mg/mL IgG (MW = 150,000) is needed for labeled protein. Then, dissolve a tube containing 1 mg of CY dye in 100 μL of DMSO. This means that 3.95 μL of CY volume is needed. Using CY3-NHS ester as an example, the calculation process is as follows in detail: 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) μL (CY3-NHS ester) = mmol (CY3-NHS ester) ×MW (CY3-NHS ester) / mg/μL (CY3-NHS ester) = 6.7×10-5 mmol× 590.15 mg/mmol / 0.01 mg/μL = 3.95 μL (CY3-NHS ester) Usage method 1. First, label the reaction by adding 0.5 mL of the protein sample solution to the calculated volume of fresh carrier containing 10 mg/mL CY dye. Gently shake the mixture to combine, and then quickly gather the centrifuged sample at the bottom of the reaction tube. 2) Do not copy: Place the reaction tube in a dark area, give it a gentle shake, and walk for 60 minutes in the initial conditions. You should also gently flip the reaction tube over several times to 2. Using SepHadex G-25 column blocked dye conjugates as an example, the protocol that follows involves protein blocking and desalting. 1) Prepare the SepHadex G-25 column as directed by the manufacturer. 2) Fill the SepHadex G-25 column to the brim with the reaction mixture. PBS (pH 7.2–7.4) should be added when the sample dips below the top resin's surface. 4) To finish columnar shrinkage, immediately add more PBS (pH 7.2–7.4) to the intended sample. Mix the ingredients that contain the chosen protein-dye combination.
In vitro, Cy5 maleimide is a thiol-reactive cyanine-based fluorescent reagent for covalent labeling of cysteine residues in proteins and peptides. It delivers red/NIR fluorescence for imaging and fluorescence-based assays. The dye is used in flow cytometry, and other labeling techniques. It is a highly sensitive cyanine dye that can be used to detect free thiols in native protein extracts. Its long wavelength, adjustable absorption and emission, high extinction coefficient, and good water solubility are characteristic of cyanine dyes.

In vivo activity of Cy5 maleimide is not typically studied, as it is a labeling reagent. However, Cy5-labeled proteins or peptides can be used in vivo for imaging applications. The red/near-infrared fluorescence of Cy5 allows for deep tissue imaging with reduced background autofluorescence. This makes it a valuable tool for studying protein distribution, tumor targeting, and other biological processes in living animals.

In vitro enzyme/receptor binding (non-cell) assays using Cy5 maleimide typically involve labeling a protein or peptide with the dye. A standard protocol for labeling a protein: the protein is dissolved in a buffer (e.g., PBS, pH 7.4) and treated with a reducing agent (e.g., TCEP) to reduce disulfide bonds. Cy5 maleimide is added to the protein solution, and the reaction mixture is incubated at room temperature for 1-2 hours in the dark. The labeled protein is purified by dialysis or size exclusion chromatography. The degree of labeling is determined by measuring the absorbance at 650 nm (Cy5) and 280 nm (protein).

In vitro cell-based assays using Cy5 maleimide typically involve labeling cell surface or intracellular proteins. A standard protocol: cells are harvested and incubated with a Cy5 maleimide-labeled antibody or other protein in PBS for 30-60 minutes at 4°C. Cells are washed to remove unbound probe and analyzed by flow cytometry. The fluorescence intensity measured corresponds to the expression level of the target protein. Alternatively, the labeled protein can be used for immunofluorescence microscopy to visualize the target protein.

In vivo animal experiments for Cy5 maleimide are typically not performed for the compound itself, but for Cy5-labeled biomolecules. A standard protocol for imaging a Cy5-labeled antibody: the labeled antibody is administered intravenously to mice. At various time points, animals are imaged using an in vivo imaging system equipped with appropriate excitation and emission filters for Cy5 (excitation ~650 nm, emission ~670 nm). The fluorescence signal is used to track the distribution and accumulation of the antibody in tumors or other tissues. This approach is widely used in cancer research and drug development.

Pharmacokinetic properties of Cy5 maleimide are not a primary focus of study. The compound is a reactive labeling reagent that is typically used in vitro. Once conjugated to a biomolecule, the pharmacokinetics of the conjugate are determined by the biomolecule rather than the dye. The compound is typically stored at -20°C, protected from light.

Toxicity data for Cy5 maleimide is limited. As a fluorescent dye, it is generally considered to have low toxicity at the concentrations used for labeling. However, as with all chemical reagents, appropriate safety precautions should be taken when handling the compound. Inhalation, ingestion, and skin contact should be avoided. The compound is not intended for human use.

[1]. Cy5 maleimide labelling for sensitive detection of free thiols in native protein extracts: identification of seed proteins targeted by barley thioredoxin h isoforms. Biochem J. 2004 Mar 1;378(Pt 2):497-507.

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

[3]. 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.

Cy5 maleimide is a thiol-reactive cyanine-based fluorescent dye for covalent labeling of cysteine residues. It delivers red/NIR fluorescence for imaging and assays. It is used for labeling proteins and peptides. It is not a drug and has no clinical use or approval status. It is commercially available from chemical suppliers for research purposes only.

C38H45CLN4O3

641.2419

640.318

1437796-65-0

1437872-46-2 (without anion);1437796-65-0 (chloride);

91757870

Brown to dark brown solid powder

1

5

12

46

1270

0

[Cl-].O=C(CCCCCN1C2=CC=CC=C2C(C)(C)/C/1=C/C=C/C=C/C1C(C)(C)C2=CC=CC=C2[N+]=1C)NCCN1C(C=CC1=O)=O

IATQCUIJHBSCGA-UHFFFAOYSA-N

InChI=1S/C38H44N4O3.ClH/c1-37(2)28-16-11-13-18-30(28)40(5)32(37)20-8-6-9-21-33-38(3,4)29-17-12-14-19-31(29)41(33)26-15-7-10-22-34(43)39-25-27-42-35(44)23-24-36(42)45;/h6,8-9,11-14,16-21,23-24H,7,10,15,22,25-27H2,1-5H3;1H

6-[(2E)-3,3-dimethyl-2-[(2E,4E)-5-(1,3,3-trimethylindol-1-ium-2-yl)penta-2,4-dienylidene]indol-1-yl]-N-[2-(2,5-dioxopyrrol-1-yl)ethyl]hexanamide;chloride

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 : ~125 mg/mL (~194.93 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.24 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.)