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.

[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.

C33H40N2O8S2

656.81

656.222

146368-11-8

91811031

Brown to green black solid powder

7.515

2

9

11

45

1430

0

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

HPICMEGAGMPYID-UHFFFAOYSA-N

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)

(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

Sulfo Cy5 acid Sulfo-Cy5 acid Sulfo-Cy5-acid

2934.99.9001

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.

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

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.

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)