ICG Maleimide 

ICG Maleimide is thiol reactive near infrared (NIR) fluorescent dye and used to generate a stable fluorescence signal in bioimaging (Ex/Em = 789/813 nm).

Protocol
1.Protein Preparetion
1) In order to obtain the best labeling effect, please prepare the protein (antibody) concentration as 2 mg/mL.
2) The pH value of protein solution shall be 6.5±0.5.
3) If the protein concentration is lower than 2 mg/mL, the labeling efficiency will be greatly reduced. In order to obtain the best labeling efficiency, it is recommended that the final protein concentration range is 2-10 mg/mL.
2.Dye Preparation
Add anhydrous DMSO into the vial of ICG Maleimideto make a 10 mM stock solution. Mix well by pipetting or vortex.
3.Calculation of dye dosage
The amount of ICG Maleimide required for reaction depends on the amount of protein to be labeled, and the optimal molar ratio of ICG Maleimide to protein is about 10.
Example: The concentration of the protein is ~0.05 mM assuming the protein concentration is 10 mg/mL and the molecular weight of the protein is ~200 Kda. 4.Run conjugation reaction
1) Add 5 µL of the dye stock solution (10 mM) into the vial of the protein solution (95 µL) with effective shaking.
In solution, gently shake to mix, then centrifuge briefly to collect the sample at the bottom of the reaction tube. Don'tmix well to prevent protein samples from denaturation and inactivation.
2) The reaction tubules were placed in a dark place and incubated gently at room temperature for 60 minutes at intervals.For 10-15 minutes, gently reverse the reaction tubules several times to fully mix the two reactants and raise the bar efficiency.
5.Purify the conjugation
The following protocol is an example of dye-protein conjugate purification by using a SepHadex G-25 column.
1) Prepare SepHadex G-25 column according to the manufacture instruction.
2) Load the reaction mixture (From "Run conjugation reaction") to the top of the SepHadex G-25 column.
3) Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
4) Add more PBS (pH 7.2-7.4) to the desired sample to complete the column purification. Combine the fractions that contain the desired dye-protein conjugate.

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

853.08

C₅₁H₅₆N₄O₆S

2143933-81-5

Solid

Green to dark green

O=C(NCCN1C(C=CC1=O)=O)CCCCCN(C(C=CC2=C3C=CC=C2)=C3C/4(C)C)C4=C/C=C/C=C/C=C/C(C5(C)C)=[N+](CCCCS(=O)([O-])=O)C6=C5C7=C(C=C6)C=CC=C7

Room temperature in continental US; may vary elsewhere.

4°C, protect from light

*In solvent : -80°C, 6 months; -20°C, 1 month (protect from light)

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (protect from light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

• [1]. Johansen ML,et al. Comparison of Near-Infrared Imaging Agents Targeting the PTPmu Tumor Biomarker. Mol Imaging Biol. 2023 Aug;25(4):744-757.  [Content Brief]