Ac-LEHD-AFC

Ac-LEHD-AFC is a fluorescent substrate for caspase-9, can be used to assess the activity of caspase^[1]. IC50 & Target: Caspase-9^[1] In Vitro: Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs).
The assessment of the caspase activity by fluorogenic substrate assay^[1]:
1. Preparation of cell lysis buffer: 1 % Nonidet P-40, NaCl 200 mM, Tris/HCl 20 mM, pH 7.4, Leupeptin 10 mg/L, Aprotinin (HY-P0017) (0.27 kU/L) and 100 mM PMSF (HY-B0496) .
2. Incubate the lysis buffer (25 mg of total protein) with 50 mmol/L of AC-DEVD-AMC (HY-P1003) (caspase-3), AC-VEID-AFC (caspase-8) or AC-LEHD-AFC (caspase-9) in the buffer (HEPES 10 mM, pH 7.4, containing Mannitol 220 mM, Sucrose 68 mM, NaCl 2 mM, KH₂PO₄ 2.5 mM, EGTA 0.5 mM, MgCl₂ 2 mM, Pyruvate 5 mM, PMSF 0.1 mM, and Dithiothreitol 1 mM).
3. Assess the caspase activity by measuring fluorescent 7-amino-4-methylcoumarin released for 1 h at a 2-min intervals by a spectrofluorometer.