Suc-Leu-Leu-Val-Tyr-AMC (solution) 

Suc-Leu-Leu-Val-Tyr-AMC (solution) is a membrane-permeable calpain-specific fluorogenic substrate (Ex/Em = 390/480 nm)^[1].
Solution Concentration: 20 mM

1. Solution preparation^[1]
1.1 Stock solution
Storage: Store at -20°C or -80°C in dark after aliquoting. Avoid repeated freezing and thawing.
1.2 Preparation of working solution
Dilutewith assay buffer (optimized according to the experiment). The corresponding stock solution can be diluted according to the actual situation. Note that if the solvent is DMSO, the cytotoxicity of DMSO must be considered, and a solvent control should be prepared; if the solvent is pure water, the working solution needs to be filtered and sterilized before adding cells.
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].

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

763.89

C₄₀H₅₃N₅O₁₀

94367-21-2

CC(C1=CC=C(NC([C@@H](NC([C@H](C(C)C)NC([C@H](CC(C)C)NC([C@H](CC(C)C)NC(CCC(O)=O)=O)=O)=O)=O)CC2=CC=C(O)C=C2)=O)C=C1O3)=CC3=O

Room temperature in continental US; may vary elsewhere.

Please store the product under the recommended conditions in the Certificate of Analysis.

• [1]. Roberta L. Debiasi, et al. Reovirus-Induced Apoptosis Is Preceded by Increased Cellular Calpain Activity and Is Blocked by Calpain Inhibitors. J Virol. 1999 Jan; 73(1): 695-701.  [Content Brief]

  [2]. Tadlock L, et al. Transforming growth factor-beta inhibition of proteasomal activity: a potential mechanism of growth arrest. Am J Physiol Cell Physiol. 2003 Aug;285(2):C277-85. Epub 2003 Mar 19.  [Content Brief]

  [3]. Gardner RC, et al. Characterization of peptidyl boronic acid inhibitors of mammalian 20 S and 26 S proteasomes and their inhibition of proteasomes in cultured cells. Biochem J. 2000 Mar, 2:447-54.  [Content Brief]