CY5 is a novel and potent photoswitchable fluorescent labeling reagent and reactive dye used for labeling of amino-groups in peptides, proteins, and oligonucleotides. It requires small amount of organic co-solvent (such as DMF or DMSO) to be used in labeling reaction. This reagent is ideal for very cost-efficient labeling of soluble proteins, as well as all kinds of peptides and oligonucleotides. This reagent also works well in organic solvents for small molecule labeling.Excitation (nm):649, Emission (nm): 670.

Physicochemical Properties

Molecular Formula	C33H40N2O8S2
Molecular Weight	656.81
Exact Mass	656.222
CAS #	146368-11-8
PubChem CID	91811031
Appearance	Brown to green black solid powder
LogP	7.515
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	9
Rotatable Bond Count	11
Heavy Atom Count	45
Complexity	1430
Defined Atom Stereocenter Count	0
SMILES	CCN\1C2=C(C=C(C=C2)S(=O)(=O)[O-])C(/C1=C/C=C/C=C/C3=[N+](C4=C(C3(C)C)C=C(C=C4)S(=O)(=O)O)CCCCCC(=O)O)(C)C
InChi Key	HPICMEGAGMPYID-UHFFFAOYSA-N
InChi Code	InChI=1S/C33H40N2O8S2/c1-6-34-27-18-16-23(44(38,39)40)21-25(27)32(2,3)29(34)13-9-7-10-14-30-33(4,5)26-22-24(45(41,42)43)17-19-28(26)35(30)20-12-8-11-15-31(36)37/h7,9-10,13-14,16-19,21-22H,6,8,11-12,15,20H2,1-5H3,(H2-,36,37,38,39,40,41,42,43)
Chemical Name	(2Z)-2-[(2E,4E)-5-[1-(5-carboxypentyl)-3,3-dimethyl-5-sulfoindol-1-ium-2-yl]penta-2,4-dienylidene]-1-ethyl-3,3-dimethylindole-5-sulfonate
Synonyms	Sulfo Cy5 acid Sulfo-Cy5 acid Sulfo-Cy5-acid
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: This product requires protection from light (avoid light exposure) during transportation and storage.
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 finest preparation of stock solution 1. Protein preparation To get the labeling effect, add protein (antibody) at a concentration of 2 mg/mL. 1) The pH value of the protein solution should be 8.5±0.5. If the pH is lower than 8.0, use 1 M carbon dioxide. 2) If the protein content is lower than 2 mg/mL, the labeling efficiency will be considerably lowered. In order to acquire the best labeling efficiency, it is recommended that conclude Protein concentration range is 2-10 mg/mL. 3) The protein must be in a clear buffer containing primary amines (such as Tris or glycine) and ammonium ions, else the labeling efficiency will be impaired. 2. Dye preparation Add anhydrous DMSO to CY dye to generate a 10 mM stock solution. Mix well by glass tube or vortex. The CY storage solution is recommended to be stored in -20℃ or -80℃ in the dark after being aliquoted. 3. Amount of dye working solution The amount of CY dye required for the labeling reaction depends on the amount of labeled protein. The ideal dosage of CY dye and protein is as follows: Assume that the needed labeled protein is 500 μL of 2 mg/mL IgG (MW =150,000), use 100 μL DMSO to dissolve a tube of 1 mg CY dye, then the required CY volume is 3.95 μL. In detail, the calculation process is as follows (taking CY3-NHS ester as an example): 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. Labeling reaction 1) Take the calculated volume of fresh carrier of 10 mg/mL CY dye slowly Add to 0.5 mL protein sample solution, shake gently to mix, and briefly collect the centrifuged sample at the bottom of the reaction tube. Do not imitate 2) Set the reaction tube in a dark area, shake it gently and walk for 60 minutes under the original conditions, 10-15 minutes per week, gently turn the reaction tube over many times to 2. Protein blocking and desalting The following approach is to employ SepHadex G-25 column blocked dye conjugates as an example. 1) Prepare SepHadex G-25 column according to the manufacturer's instructions. 2) Insert the reaction mixture into the top of the SepHadex G-25 column. 3) When the sample runs below the surface of the top resin, add PBS (pH 7.2-7.4). 4) Immediately add extra PBS (pH 7.2-7.4) to the chosen sample to finish columnar shrinking. Combine the components comprising the appropriate dye-protein conjugate.

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 (~190.31 mM) H2O : ~25 mg/mL (~38.06 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.17 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 (3.17 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	1.5225 mL	7.6126 mL	15.2251 mL
	5 mM	0.3045 mL	1.5225 mL	3.0450 mL
	10 mM	0.1523 mL	0.7613 mL	1.5225 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.