Proteasome Activity Assay in Muscle

Frozen muscles were homogenized using a homogenizer in a proteasome assay buffer. The proteasome assay buffer contained 50 mM Tris-HCl (pH 7.5), 2 mM ATP, 1 mM DTT (dithiothreitol), 150 mM NaCl, 5 mM MgCl₂, and 10% glycerol. The extracts were centrifuged at 12,000 rpm for 30 minutes at 4 °C and the supernatant was collected. Protein concentrations were determined using the Lowry Protein Assay (Multiskan FC microplate photometer, Thermo Fisher Scientific). Proteasome activity was assessed using the Proteasome-Glo Assay kit (Promega) following the manufacturer's instructions. Chymotrypsin-like activity assays were conducted using quadriceps muscle homogenates in a white 96-well plate, according to a procedure described in detail elsewhere (8 (link)).

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Li Q., Ishii K.A., Kamoshita K., Takahashi K., Abuduwaili H., Takayama H., Galicia-Medina C.M., Tanida R., Ko Oo H., Gafiyatullina G., Yao X., Abuduyimiti T., Hamazaki J., Goto H., Nakano Y., Takeshita Y., Harada K., Murata S, & Takamura T. (2023). PAC1 Deficiency Protects Obese Male Mice From Immobilization-Induced Muscle Atrophy by Suppressing FoxO–Atrogene Axis. Endocrinology, 164(6), bqad065.

Publication 2023

Proteasome-Glo Assay kit

Assays Buffer Chymotrypsin Dithiothreitol Frozen Glycerol Mgcl2 Muscles Nacl Proteasome Protein Quadriceps muscle Tris

Corresponding Organization : Kanazawa University

Other organizations : National Center for Geriatrics and Gerontology, Kanazawa Medical University, The University of Tokyo

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Variable analysis

independent variables

Proteasome assay buffer composition (50 mM Tris-HCl (pH 7.5), 2 mM ATP, 1 mM DTT, 150 mM NaCl, 5 mM MgCl2, 10% glycerol)

dependent variables

Proteasome activity (chymotrypsin-like activity)
Protein concentration

control variables

Centrifugation conditions (12,000 rpm for 30 minutes at 4 °C)
Lowry Protein Assay for determining protein concentrations
Proteasome-Glo Assay kit for measuring proteasome activity

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