Western Blot Analysis of OXPHOS Proteins

Western blot analyses were performed in Bioplex‐lysates of human muscle tissue as previously described (Wefers et al., 2020 (link)). Equal amounts of proteins were loaded on gradient Bolt 4%–12% gels (Novex, Thermo Fisher Scientific). Proteins were transferred to nitrocellulose with the Trans‐Blot Turbo transfer system (Bio‐Rad Laboratories). The following antibodies and dilutions were used in this study: a cocktail of mouse monoclonal antibodies directed against human OXPHOS (dilution 1:5.000; ab110411, Abcam), as well as antibodies directed against TOMM20 (dilution 1:10.000; aba186734; Abcam), porin/VDAC (dilution 1:1.000; sc‐390,996; 1:5000, Santa Cruz biotechnology). The specific proteins were detected using secondary antibodies conjugated with IRDye680 or IRDye800 and were quantified with the CLx Odyssey Near Infrared Imager (Li‐COR, Westburg).

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Mancilla R., Pava‐Mejia D., van Polanen N., de Wit V., Bergman M., Grevendonk L., Jorgensen J., Kornips E., Hoeks J., Hesselink M.K, & Schrauwen‐Hinderling V.B. (2023). Invasive and noninvasive markers of human skeletal muscle mitochondrial function. Physiological Reports, 11(12), e15734.

Publication 2023

Novex Trans‐Blot Turbo transfer system ab110411 sc‐390,996 CLx Odyssey Near Infrared Imager

Antibodies Antibodies cocktail Bioplex Dilution Gels Human Irdye800 Mouse Muscle tissue Nitrocellulose Porin vdac Proteins Tomm20 Western blot analyses

Corresponding Organization : German Center for Diabetes Research

Other organizations : Finis Terrae University

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Variable analysis

independent variables

Manipulation of human muscle tissue samples

dependent variables

Protein expression levels of OXPHOS, TOMM20, and porin/VDAC as measured by Western blot analysis

control variables

Equal amounts of proteins loaded on gradient Bolt 4%–12% gels
Proteins transferred to nitrocellulose with the Trans‐Blot Turbo transfer system
Specific proteins detected using secondary antibodies conjugated with IRDye680 or IRDye800 and quantified with the CLx Odyssey Near Infrared Imager

controls

Positive control: Cocktail of mouse monoclonal antibodies directed against human OXPHOS
Negative control: Not explicitly mentioned

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