Suc-Leu-Leu-Val-Tyr-AMC

Suc-Leu-Leu-Val-Tyr-AMC is a membrane-permeable calpain-specific fluorogenic substrate (Ex/Em = 390/480 nm)^[1]. In Vitro: 1. Solution preparation^[1]
1.1 Preparation of stock solution
Solvent: DMSO
Concentration: 62.5 mM is recommended.
Storage: Store at -20°C or -80°C in dark after aliquoting. Avoid repeated freezing and thawing.
1.2 Preparation of working solution
Dilute to 6.25 µM with assay buffer (optimized according to the experiment).
Note: The working solution should be prepared and used immediately. Keep it away from light.

2. Cell staining
2.1 L929 cells are plated at 10⁵ cells in 200 μL per well in 96-well plates.
2.2 Add 200 μL of assay buffer (115 mM NaCl, 1 mM KH₂PO₄, 5 mM KCl, 2 mM CaCl₂, 1.2 mM MgSO₄, 25 mM sodium HEPES buffer (pH 7.4)) to all wells.
2.3 Add Suc-Leu-Leu-Val-Tyr-AMC (62.5 µM) to the cells.
2.4 The plates are incubated at 37°C in the fluorometer, and fluorescence was measured at 5-min intervals starting immediately after the addition of substrate.

Suc-Leu-Leu-Val-Tyr-AMC (Suc-LLVY) is a membrane-permeable calpain-specific fluorogenic substrate, pteolytic hydrolysis of the peptidyl-7-amino bond liberates the highly fluorescent 7-amino-4-methylcoumarin (AMC) moiety^[1]. The effect of TGF-β on hydrolysis of these substrates (e.g Suc-Leu-Leu-Val-Tyr-AMC) are assessed. Biliary epithelial H69 cells are incubated with 10, 1, 0.1, or 0 ng/mL TGF-β for 24 h. Substrate hydrolysis is then fluorometrically assessed in cytosolic extracts. Basal activity is 1.12, 8.33, and 14.52 nmol AMC/mg protein/min for suc-LLVY-AMC, z-LLE-AMC, and z-LLL-AMC hydrolysis, respectively^[2].