Cy2 Iodide (solution)

Guidelines (The following is our recommended protocol. This protocol is provided for guidance only and should be modified according to your specific needs.)
1. Protein Preparation
To achieve optimal labeling results, prepare the protein (antibody) at a concentration of 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 using 1 M sodium bicarbonate.
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 of 2–10 mg/mL is recommended.
1.3 The protein must be in a buffer that is 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 first be activated with Condensation Solution (500 μg/mL) (HY-D0178) before proceeding with the subsequent labeling experiment.
2. Calculation of Dye Amount
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. Note: We recommend using a 10:1 dye-to-protein molar ratio. If the concentration is too low or too high, the ratio can be adjusted to 5:1, 15:1, or 20:1.
3. Perform the Conjugation Reaction
1. Slowly add the calculated volume of freshly prepared 10 mg/mL dye to 0.5 mL of protein sample solution. Gently shake to mix, then briefly centrifuge to collect the sample at the bottom of the reaction tube. Avoid vigorous mixing to prevent denaturation and inactivation of the protein sample.
2. Place the reaction tube in the dark and gently shake-incubate at room temperature for 60 minutes. Every 10–15 minutes, gently invert the reaction tube several times to thoroughly mix the reactants and improve labeling efficiency.
4. Purification of the Conjugate
The following protocol is an example of using the Labelling Kits Centrifugation-Based Rapid Desalting Column (5KD) (HY-D3014) to purify the dye-protein conjugate.
4.1 Prepare the desalting column according to the instructions.
4.2 Load the reaction mixture onto the top of the desalting column.
4.3 Once the sample has run below the resin surface, immediately add PBS (pH 7.2–7.4).
4.4 Add more PBS (pH 7.2–7.4) to the target sample to complete the column purification. Collect the fractions containing the desired dye-protein conjugate.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.