Creating Optimal Western Blot Conditions for OPA1 Isoforms in Skeletal Muscle Cells and Tissue

Current protocols Pub Date : 2025-02-05 DOI:10.1002/cpz1.70004

Margaret Mungai, Amber Crabtree, Han Le, Johnathan Moore, Desiree Nguyen, Benjamin Rodriguez, Chanel Harris, Dominique C. Stephens, Heather K. Beasley, Edgar Garza-Lopez, Kit Neikirk, Bryanna Shao, Ashton Oliver, Genesis Wilson, Serif Bacevac, Larry Vang, Zer Vue, Neng Vue, Andrea G. Marshall, Kyrin Turner, Elma Zaganjor, Jianqiang Shao, Sandra Murray, Jennifer A. Gaddy, Celestine Wanjalla, Jamaine Davis, Steven M. Damo, Lori D. Banks, Antentor Hinton Jr

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Abstract

OPA1 is a dynamin-related GTPase that modulates mitochondrial dynamics and cristae integrity. Humans carry eight different isoforms of OPA1 and mice carry five, all of which are expressed as short- or long-form isoforms. These isoforms contribute to OPA1's ability to control mitochondrial energetics and DNA maintenance. However, western blot isolation of all long and short isoforms of OPA1 can be difficult. To address this issue, we developed an optimized western blot protocol based on improving running time to isolate five different isoforms of OPA1 in mouse cells and tissues. This protocol can be applied to study changes in mitochondrial structure and function. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Western Blot Protocol for Isolating OPA1 Isoforms in Mouse Primary Skeletal Muscle Cells

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