Protocol 1: Preparing proteins 1) Please prepare the protein (antibody) concentration to 2 mg/mL in order to achieve the greatest labeling effect. 2) The protein solution has a pH of 8.5±0.5. One milligram of sodium bicarbonate (1M) should be added to the pH if it is less than 8.0. 3) The labeling efficiency will be significantly decreased if the protein content is less than 2 mg/mL. The range of suggested final protein concentration is 2–10 mg/mL for best labeling efficiency. 4) To ensure optimal labeling efficacy, the protein needs to be in a buffer free of ammonium ions and primary amines, like Tris or glycine. 2. Dye preparation (using, for instance, CY3-NHS ester) To create a 10 mM stock solution, fill the CY3-NHS ester vial with anhydrous DMSO. Use a pipette or vortex to thoroughly mix. 3. The dosage calculation for dye. The amount of protein to be labeled determines how much CY3-NHS ester is needed for the reaction; the ideal molar ratio for CY3-NHS is roughly 10. For instance: Assuming that 500 μL 2 mg/mL IgG (MW=150,000) is the necessary marker protein, dissolve 1 mg of CY3-NHS ester in 100 μL DMSO to get the 5.05 μL of CY3-NHS ester that is needed. The following is the precise calculating procedure: 1) mg/mL(IgG) ×mL(IgG)/MW(IgG) = 2 mg/mL × 0.5 mL/150,000 mg/mmol = 6.7×10-6 mmol 2) moles(CY3-NHS ester) = moles(IgG) × 10=6.7 × 10-6 mmol×10=6.7 × 10-5 mmol 3) uL(CY3-NHS ester) = mmol(CY3-NHS 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. Start the coupling reaction. 1) Add the required quantity of recently made CY3-NHS ester (10 mg/mL). A 0.5 mL protein sample should be added gradually to the solution, gently shaken to combine, and then quickly centrifuged to gather the material at the reaction tube's bottom. To prevent denaturation and inactivation of protein samples, do not mix them equally. 2) After placing the reaction tube in a dark location, gently incubate it for 60 minutes at room temperature. After 10 to 15 minutes, gently reverse the response. 5. Make the conjugate pure An example of a SepHadex G-25 column purification process for a dye-protein conjugate is provided below. br/> First, 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 (from "Run conjugation reaction"). 3) Add PBS (pH 7.2–7.4) as soon as the sample is running below the top resin surface. To finish the purification of the chosen sample, add additional PBS (pH 7.2–7.4). The appropriate dye-protein conjugate-containing fractions were combined.

[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

C45H60N4O10S2

881.11

CY7-SE;477908-53-5

Typically exists as solids (or liquids in special cases) at room temperature

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.  (2). This product is not stable in solution, please use freshly prepared working solution for optimal results.

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

DMSO :~83.33 mg/mL (~94.57 mM)
H2O :~10 mg/mL (~11.35 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (2.36 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 (2.36 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.)