Boc-Lys-AMC

Enzyme activity assay^[1]

This plan is based on literature, please modify it to meet your specific needs.

1. Preparation of substrate working solution

(1) Prepare storage solution: Dissolve Boc-Lys-AMC in DMSO at concentration of 10-50mM.

Note: Unused storage solution should be aliquoted and stored in the dark at -20°C or -80°C to avoid repeated freezing and thawing.

(2) Prepare working solution: Dilute the storage solution with a reaction buffer.

Note: Prepare the working solution fresh and store briefly at 4°C or on ice.

2. Preparation of enzyme working solution: Dilute enzyme with reaction buffer.

3. Prepare a 96-well plate:

Reaction wells: Add a mixture of Boc-Lys-AMC substrate and enzyme at concentrations of 10-50μM and 5-50nM, respectively.

Blank wells: Add Boc-Lys-AMC substrate at a concentration of 10-50μM, add reaction buffer to make up the volume.

4. Pre-incubate the 96-well plate at 37℃ for 10-60min.

5. Measure the fluorescence intensity at 440-460nm under 350-360nm excitation using a fluorescence spectrophotometer.

Precautions:

1) Fluorescent substrate has 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.

References:
[1] Ueki N, Wang W, Swenson C, McNaughton C, Sampson NS, Hayman MJ. Synthesis and Preclinical Evaluation of a Highly Improved Anticancer Prodrug Activated by Histone Deacetylases and Cathepsin L. Theranostics. 2016;6(6):808-816.