A protocol is given at the MedChemExpress web page for the product. The protocol is for an amine-reactive NHS ester (such as the related dyes HY-172286 or HY-D1568), however, not the carboxylic acid shown in the picture on the page for HY-D0825. To couple the acid to amine groups in the protein would require using a carbodiimide, such as EDC. See below for a link to using EDC.

https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/carbodiimide-crosslinker-chemistry.html#2

Hello, Marine Ma

Before use, add 50 μL of 500 μg/mL condensation solution to approximately 10 μL of the mother solution for activation.

If you need detailed Protocols: Adam B Shapiro also mentioned the relevant link.

Stock solution preparation

1. Protein preparation

For optimal labeling, prepare the protein (antibody) concentration to 2 mg/mL.

1) The pH of the protein solution should be 8.5±0.5. If the pH is lower than 8.0, adjust with 1 M sodium bicarbonate.

2) If the protein concentration is lower than 2 mg/mL, the labeling efficiency will be greatly reduced. For optimal labeling efficiency, the recommended final protein concentration range is 2-10 mg/mL.

3) The protein must be in a buffer free of primary amines (such as Tris or glycine) and ammonium ions, otherwise the labeling efficiency will be affected.

2. Dye preparation

Dilute CY dye in anhydrous DMSO to make a 10 mM stock solution. Mix thoroughly by glass tube or vortex.

Note: It is recommended that the CY stock solution be stored at -20 ℃ or -80 ℃ in the dark after aliquoting.

Before use, the condensation solution (500 μg/mL) (HY-D0178) must be used for activation before subsequent labeling experiments can be performed.

3. Calculation of the amount of dye working solution

The amount of CY dye required for the labeling reaction depends on the amount of protein to be labeled. The optimal molar ratio of CY dye to protein is about 10.

For example: If the required labeled protein is 500 μL of 2 mg/mL IgG (MW=150,000), and 100 μL DMSO is used to dissolve a tube of 1 mg CY dye, the required CY volume is 3.95 μL. The detailed 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)

Instructions

1. Labeling reaction

1) Take a calculated volume of freshly prepared 10 mg/mL CY dye and slowly add it to 0.5 mL of protein sample solution, gently shake to mix, and then briefly centrifuge to collect the sample at the bottom of the reaction tube. Avoid violent mixing to prevent protein sample denaturation and inactivation.

2) Place the reaction tube in a dark place, gently shake and incubate at room temperature for 60 minutes. Every 10–15 minutes, gently invert the reaction tube several times to fully mix the two reactants and improve labeling efficiency.

2. Protein purification and desalting

The following scheme uses SepHadex G-25 column to purify dye-protein conjugates as an example.

1) Prepare SepHadex G-25 column according to the manufacturer's instructions.

2) Load the reaction mixture onto the top of the SepHadex G-25 column.

3) As soon as the sample runs below the top resin surface, add PBS (pH 7.2-7.4).

4) Add more PBS (pH 7.2-7.4) to the desired sample to complete the column purification. Combine the fractions containing the desired dye-protein conjugate.

Details and Protocol can be found at the following link:

https://www.medchemexpress.com/CY7.html

The answer to this question comes from MedChemExpress (MCE) Technical Support.

[email protected]