Lung ACE2 Protein Quantification

Lung proteins were extracted with TNE lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% NP40, protease inhibitor (Complete Mini; Roche) using Microsmash (MS-100R; TOMY), 20 mM NaF, 2 mM Na₃VO₄), were sonicated and denatured with LDS sample buffer (Invitrogen) at 70°C. Proteins were electrophoresed on NuPAGE bis-tris precast gels (Invitrogen) and transferred to nitrocellulose membranes (Invitrogen). Membranes were probed with anti-mouse ACE2 antibody [20 (link)] and anti-mouse GAPDH antibody (Cell Signaling TECHNOLOGY, 14C10). The blotting bands visualized with ECL reagent (Bio-Rad) using ChemiDoc Touch Imaging System (Bio-Rad). Image Lab software was used to quantify band intensity.

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Minato T., Yamaguchi T., Hoshizaki M., Nirasawa S., An J., Takahashi S., Penninger J.M., Imai Y, & Kuba K. (2022). ACE2-like enzyme B38-CAP suppresses abdominal sepsis and severe acute lung injury. PLoS ONE, 17(7), e0270920.

Publication 2022

Complete Mini (MS-100R; LDS sample buffer NuPAGE bis-tris precast gels nitrocellulose membranes anti-mouse GAPDH antibody ECL reagent ChemiDoc Touch Imaging System

Ace2 Anti antibody Bis tris Buffer Edta Gapdh Gels Lung Membranes Mouse Nacl Nitrocellulose Protease inhibitor Proteins Touch Tris

Corresponding Organization : University of British Columbia

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Variable analysis

independent variables

Lung proteins extracted with TNE lysis buffer
Sonication
Denaturation with LDS sample buffer at 70°C

dependent variables

Expression of ACE2 protein
Expression of GAPDH protein

control variables

50 mM Tris
150 mM NaCl
1 mM EDTA
1% NP40
Protease inhibitor (Complete Mini; Roche)
20 mM NaF
2 mM Na3VO4

controls

Anti-mouse GAPDH antibody (Cell Signaling TECHNOLOGY, 14C10) as a loading control

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