AF488 NHS ester

Detection method based on AF488 NHS ester

This protocol only provides a guideline, and should be modified according to your specific needs

1. Prepare dyeing solution

(1) Prepare stock solution: Prepare AF488 NHS ester stock solution with a concentration of 1mM in high-quality anhydrous DMSO. Notice: Aliquot the unused stock solution and store it in the dark at -20°C to avoid repeated freezing and thawing.

(2) Prepare working solution: Dilute the stock solution with a suitable buffer (such as PBS) to prepare a working solution with a concentration of 1μM. Please adjust the concentration of the working fluid according to the actual situation, and prepare it now to avoid repeated freezing and thawing. Notice: The working solution must be prepared fresh on the day of the experiment.

2. Protein Preparation

(1) Exchange protein into 0.1M NaHCO₃ buffer (pH 8.3) using a spin filter or desalting column.

(2) Measure absorbance at 280nm and calculate concentration and mix AF488 NHS ester and protein at a 3:1 molar ratio (dye:protein) to achieve=0.8-1.0 dye per protein.

(3) Incubate 1h at room temperature in the dark with gentle agitation.

3. Quenching and detection

(1) Add 10µl 1M Tris pH 8.0 per 100µl reaction volume and incubate 15min.

(2) Remove unreacted dye by gel-filtration or spin desalting pre-equilibrated with PBS, and measure by flow cytometry.

4. Precautions:

(1) Fluorescent dyes all have quenching problems. Please avoid light as much as possible to slow down fluorescence quenching.

(2) For your safety and health, please wear a lab coat and disposable gloves when operating

(3) AF488 NHS ester should be stored in the dark throughout the experiment.

(4) Keep reaction pH 8.0–8.5; lower pH slows coupling, higher pH increases hydrolysis

(5) This protocol and all reagents are for scientific research use only.

References:
[1] Wang J, Bhattacharyya J, Mastria E, et al. A quantitative study of the intracellular fate of pH-responsive doxorubicin-polypeptide nanoparticles[J]. Journal of Controlled Release, 2017, 260: 100-110.