Cy5 maleimide 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	C38H45CLN4O3
Molecular Weight	641.2419
Exact Mass	640.318
CAS #	1437796-65-0
Related CAS #	1437872-46-2 (without anion);1437796-65-0 (chloride);
PubChem CID	91757870
Appearance	Brown to dark brown solid powder
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	12
Heavy Atom Count	46
Complexity	1270
Defined Atom Stereocenter Count	0
SMILES	[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
InChi Key	IATQCUIJHBSCGA-UHFFFAOYSA-N
InChi Code	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
Chemical Name	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
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

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.

References

[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.

Solubility Data

Solubility (In Vitro)

DMSO : ~125 mg/mL (~194.93 mM)

Solubility (In Vivo)

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.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.5595 mL	7.7974 mL	15.5948 mL
	5 mM	0.3119 mL	1.5595 mL	3.1190 mL
	10 mM	0.1559 mL	0.7797 mL	1.5595 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.