7-Amino-4-methylcoumarin-3-acetic acid 

7-Amino-4-methylcoumarin-3-acetic acid (AMCA) serves as the parent structure of coumarin-based blue fluorescent dyes, and its activated form AMCA-NHS can directly conjugate with proteins (Ex/Em ≈ 350/440-460).

Guide (The following is our recommended protocol, which serves only as a guideline and should be modified according to your specific needs)^[1][2].
1. Conjugation Reaction
1.1 Protein Preparation: Dilute the protein to be labeled with conjugation buffer (0.5 M borate, pH 8.5) to a concentration of approximately 1-10 mg/mL. Ensure that the protein solution contains no primary amines (such as Tris, glycine, ammonium ions) to avoid competitive reactions with AMCA-NHS.
1.2 Dye Preparation: Freshly prepare an AMCA-NHS stock solution (e.g., 10 mg/mL) with anhydrous DMSO under light-protected conditions. Calculate the required amount of AMCA-NHS; a molar ratio of dye to protein between 5:1 and 20:1 is generally recommended to achieve moderate labeling.
1.3 Mixing and Reaction: Add the calculated volume of AMCA-NHS/DMSO solution dropwise to the protein solution under continuous gentle stirring or inversion mixing. Ensure that the final volume fraction of DMSO does not exceed 5-10% to prevent protein denaturation.
1.4 Incubation: Incubate the reaction mixture at room temperature (approximately 25°C) for 1-2 hours under light-protected conditions.
2. Purification and Desalting
2.1 Termination of Reaction: After incubation, excess glycine (final concentration ~50 mM) can be added to quench unreacted dye, but direct purification is generally sufficient.
2.2 Removal of Free Dye: Load the reaction mixture onto a pre-equilibrated Sephadex G-25 or G-50 gel filtration column with PBS or the desired buffer. Collect the first eluted colored protein peak (i.e., AMCA-protein conjugate) and discard the later eluted free dye peak.
2.3 Dialysis: As an alternative or supplementary step, dialyze the reaction solution against a large volume of PBS (or other suitable buffer) at 4°C overnight to completely remove small molecule impurities and salt ions.
3. Quantitative Analysis
Use a spectrophotometer to measure the absorbance of the purified conjugate at 280 nm and 350 nm (A280 and A350).

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

233.22

C₁₂H₁₁NO₄

106562-32-7

Solid

Off-white to light brown

454

344

O=C(O)CC1=C(C)C2=CC=C(N)C=C2OC1=O

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]. Khalfan H, et al. Aminomethyl coumarin acetic acid: a new fluorescent labelling agent for proteins. Histochem J. 1986;18(9):497-499.  [Content Brief]

  [2]. Wessendorf MW, et al. A spectrophotometric method for determination of fluorophore-to-protein ratios in conjugates of the blue fluorophore 7-amino-4-methylcoumarin-3-acetic acid (AMCA). J Histochem Cytochem. 1990;38(1):87-94.  [Content Brief]