Operating Instructions (The following is our recommended operating procedure. This procedure is for reference only; please adjust it according to your specific needs.) 1. Protein Preparation: To obtain the best labeling effect, prepare the protein (antibody) concentration to 2 mg/mL. 1.1 The pH of the protein solution should be 8.5 ± 0.5. If the pH is below 8.0, adjust it with 1 M sodium bicarbonate solution. 1.2 If the protein concentration is below 2 mg/mL, the labeling efficiency will be significantly reduced. For optimal labeling efficiency, a final protein concentration range of 2-10 mg/mL is recommended. 1.3 The protein must be dissolved in a buffer solution free of primary amines (such as Tris or glycine) and ammonium ions; otherwise, the labeling efficiency will be affected. Note: Before use, the protein must be activated with a condensation solution (500 μg/mL) before subsequent labeling experiments. 2. Dye Preparation: Dilute the dye with anhydrous DMSO to prepare a clear stock solution with a concentration of 10 mg/mL. 3. Dye Dosage Calculation: The amount of dye required for the reaction depends on the amount of protein to be labeled. The optimal molar ratio of dye to protein is approximately 10:1. For example, assuming the desired labeled protein is 500 μL of IgG with a concentration of 2 mg/mL (molecular weight = 150,000), and 1 mg of dye is dissolved in 100 μL of DMSO, then the required dye volume is 2.8 μL. The detailed calculation process is as follows: 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 (dye) = mmol (IgG) × 10 = 6.7 × 10⁻⁶ mmol × 10 = 6.7 × 10⁻⁵ mmol 3) μL (dye) = mmol (dye) × MW (dye)/mg/μL (dye) = 6.7 × 10⁻⁵ mmol × 418.49 mg/mmol/0.01 mg/μL = 2.8 μL (dye) Note: It is recommended that the molar ratio of dye to protein be 10:1. If the concentration is too low or too high, the ratio can be adjusted to 5:1, 15:1 or 20:1. 4. Performing the Coupling Reaction 1) Slowly add a calculated volume of freshly prepared 10 mg/mL dye to 0.5 mL of protein sample solution, gently shake to mix, then briefly centrifuge and collect the sample at the bottom of the reaction tube. Avoid vigorous shaking to prevent denaturation or inactivation of the protein sample. 2) Place the reaction tube in a dark place and incubate gently with shaking for 60 minutes at room temperature. Every 10-15 minutes, gently invert the reaction tube several times to thoroughly mix the reactants and improve labeling efficiency. 5. Purification of the Coupling The following steps demonstrate an example of purifying dye-protein conjugates using a Labelling Kits centrifugal rapid desalting column (5KD). 5.1 Prepare the desalting column according to the instructions. 5.2 Load the reaction mixture onto the top of the desalting column. 5.3 Immediately after the sample flows over the resin surface, add PBS buffer (pH 7.2-7.4). 5.4 Add more PBS buffer (pH 7.2-7.4) to the target sample to complete column purification. Collect the fraction containing the target dye-protein conjugate.

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

[2]. Use of Time-Resolved Fluorescence To Improve Sensitivity and Dynamic Range of Gel-Based Proteomics. Anal Chem. 2016 Mar 15;88(6):3067-74.

[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

C25H27IN2O4

546.40

186205-37-8

Cy2

Orange to red solid

O=C(O)CCCCC[N+](C(C=CC=C1)=C1O2)=C2/C=C/C=C3OC(C=CC=C4)=C4N\3CC.[I-]

Cyanine2 (iodine)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: 请将本产品存放在密封且受保护的环境中（例如氮气下），避免暴露在潮湿和光照下。

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

DMSO : ~50 mg/mL (~91.51 mM; ultrasonic and warming and heat to 60°C)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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