Gly-Pro-AMC hydrobromide (Gly-Pro-AMC HCl) is a specific fluorescent dye used in cytofluorescence assays to detect Dipeptidyl peptidase IV (DPP-IV) activity.

Gly-Pro-AMC hydrobromide, at a concentration of 50 µM and under incubation conditions of 20 minutes at 37 °C, serves as a specific fluorescent substrate for the detection of DPP-IV activity[2].The fluorescence properties of Gly-Pro-AMC hydrobromide can be detected under the following conditions: excitation wavelength at 350 nm and fluorescence emission spectrum detection at 450 nm[2].

Determination of DPP-4 enzyme activity in cell lysate
a. Solution preparation: In a light-proof environment, dissolve Gly-Pro-AMC hydrobromide powder with DMSO to obtain a 25 mmol/L concentrated stock solution, and dilute it to 500 μmol/L with buffer for use. Similarly, dissolve the fluorescent standard AMC powder with DMSO to obtain a 25 mmol/L concentrated stock solution, and dilute it to 160, 80, 40 and 20 μmol/L with buffer for use.
b. Operation steps:
1. Add PBS to the cells in good culture state and scrape them with a scraper to collect them into a sterile EP tube, centrifuge at 4 ℃ and 200 g for 5 min, discard the supernatant, and add 3 mL 50 mmol/L Tris-HCl buffer to resuspend the cells.
2. Add 125 μL of the above-prepared AMC standard solution of different concentrations to each well of the 96-well plate on ice, and then add 10, 20, 30, 40 and 50 μL of cell lysate to the remaining test wells, and fill the volume to 75 μL with buffer. Finally, add 50 μL of 500 μmol/L Gly-Pro-AMC hydrobromide substrate solution to each test well to make the total volume of the system 125 μL.
3. Place the 96-well plate in a multifunctional microplate reader preheated to 37 ℃ to start the reaction, and immediately measure the relative fluorescence unit excitation wavelength (λex=380 nm, emission wavelength λem=460 nm) of each well; measure every 5 minutes in the following 30 minutes.
4. Calculate the enzyme specific activity based on the standard curve of the measurement results.