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. Utilize 1 M protein if the pH is less than 8.0. 2) A protein concentration below 2 mg/mL will result in significantly poorer labeling efficiency. It is advised to use a final protein concentration range of 2–10 mg/mL to get the highest labeling efficiency. 3) There must be obvious 2 protein. To prepare the dye, make a 10 mM stock solution in the AMCA-X-SE vial by adding anhydrous dimethylimine, 3. For the dye coating preparation procedure, the ideal molar ratio of AMCA-X-SE to cosmetics is approximately 10, depending on the quantity of cosmetics to be branded. For instance: Dissolve a tube containing 1 mg AMCA-X-SE in 100 μL of DMSO if the needed labeled protein is 500 μL of 2 mg/mL IgG (MW=150,000). The AMCA-X-SE requires 3 μL of material. The exact calculation procedure 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 (AMCA-X-SE) = millimole (IgG)×10 = 6.7×10-6 mmol×10 = 6.7×10-5 mmol3) μL (AMCA-X-SE) = Millimole (AMCA-X-SE)×MW (AMCA-X-SE) / mg/μL (AMCA-X-SE) = 6.7×10-5 mmol×443 mg/mmol / 0.01 mg/μL = 3 μL ( AMCA-X-SE) Instructions for use 1. Labeling Reaction 1: Gradually add the calculated volume of freshly fixed 10 mg/mL AMCA-X-SE to the protein sample solution in 0.5 mL increments, gently shake to combine, and then quickly centrifuge to gather the sample at the tube's bottom. 2) Don't mix the dyeing process. Establish a dark environment for the reaction tube. Shake the reagent gently for 60 minutes at the initial conditions. Gently rotate the reaction tube many times every ten to fifteen minutes to reach the full 2. Protein-sealing desalting coupling: The conjugation process that follows uses an example of blocking dye conjugates using a Sephadex G-25 column. 1) Prepare the Sephadex G-25 column as directed by the manufacturer. 2) Fill the top of the Sephadex G-25 column with the reaction mixture. 3) Add PBS (pH 7.2–7.4) as soon as the sample dips below the top resin's surface. 4) To finish columnar shrinking, add additional PBS (pH 7.2–7.4) to the necessary sample. Mix the ingredients that contain the chosen protein-dye combination. Notes 1. Light and humidity can affect AMCA-X-SE. Make the AMCA-X-SE solution right away, then throw away any leftover 2. While 20–50% glycerol will decrease labeling efficiency, low amounts of folded sodium (≤3 mM or 0.02%) or thimerosal (≤0.02 mM or 0.01%) do not appreciably interfere with protein labeling. 3. Utilize buffers that include ammonium ions or primary amines (such Tris or glycine), as these substances compete with the protein that needs to be tagged. 4. This product is not intended for use in clinical diagnosis or treatment; rather, it is solely for professional use in scientific study. 5. Please wear a lab coat and disposable gloves during the operation for your own health and safety.

[1]. Surface modification of the polyethyleneimine layer on silicone oxide film via UV radiation. Applied surface science, 2009, 255(12): 6103-6106.

C22H25N3O7

443.4498

429.19

216309-02-3

59866294

White to off-white solid powder

3.342

2

8

10

32

806

0

QYSJBOMZRLAPJR-UHFFFAOYSA-N

InChI=1S/C22H25N3O7/c1-13-15-7-6-14(23)11-17(15)31-22(30)16(13)12-18(26)24-10-4-2-3-5-21(29)32-25-19(27)8-9-20(25)28/h6-7,11H,2-5,8-10,12,23H2,1H3,(H,24,26)

(2,5-dioxopyrrolidin-1-yl) 6-[[2-(7-amino-4-methyl-2-oxochromen-3-yl)acetyl]amino]hexanoate

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)