Proteasome glotm assay kit

As described in our previous study (Kitajima et al., 2014 (link)), proteasome activity was assessed using Proteasome-Glo^TM Assay kit (Promega) following the manufacturer’s instruction. The chymotrypsin-like activity assay was conducted using skeletal muscle homogenates in a total volume of 100 ml in opaque 96-well plates. For the assays, 100 mg of protein was added to assay buffer containing 20 mM Tris-HCl (pH 7.2), 0.1 mM EDTA, 5 mM ATP, 1 mM β-mercaptoethanol, 20% glycerol and 0.04% Nonidet P40. The proteasome reagent was added separately, and 30 min later, the luminescence was recorded as relative light units on a infinite F200 pro (Tecan). Each sample was measured in triplicate.

Proteasome activity was assessed using Proteasome-Glo^TM Assay kit (Promega) following the manufacturer's instruction. The trypsin-like and chymotrypsin-like activity assays were conducted using skeletal muscle homogenates in a total volume of 100 µl in opaque 96-well plates. For the assays, 120 µg of protein was added to assay buffer containing 20 mM Tris-HCl (pH 7.2), 0.1 mM EDTA, 5 mM ATP, 1 mM β-mercaptoethanol, 20% glycerol and 0.04% Nonidet P40. The individual proteasome reagents were added separately, and 30 min later, the luminescence was recorded as relative light units on a Varioskan luminometer (Thermo Scientific). Each sample was measured in triplicate.
In addition, to evaluate proteasome activity more precisely, dual measurements with or without the addition of 30 µM of the irreversible and highly specific proteasomal inhibitor adamantine-acetyl-(6-aminohexanoyl)₃-(leucinyl)₃-vinyl-(methyl)-sulfone (AdaAhx₃L₃VS, Calbiochem, catalog number 114802) were carried out (Kessler et al., 2001 (link)).