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Linear Density Sucrose Gradients to Study Mitoribosomal Biogenesis in Tissue-Specific Knockout Mice

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Mouse Genetics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2224))

Abstract

Like bacterial and cytoplasmic ribosomes, mitoribosomes are large ribonucleoprotein complexes with molecular weights in the range of several million Daltons. Traditionally, studying the assembly of such high molecular weight complexes is done using ultracentrifugation through linear density gradients, which remains the method of choice due to its versatility and superior resolving power in the high molecular weight range. Here, we present a protocol for the analysis of mitoribosomal assembly in heart mitochondrial extracts using linear density sucrose gradients that we have previously employed to characterize the essential role of different mitochondrial proteins in mitoribosomal biogenesis. This protocol details in a stepwise manner a typical mitoribosomal assembly analysis starting with isolation of mitochondria, preparation and ultracentrifugation of the gradients, fractionation and ending with SDS-PAGE, and immunoblotting of the gradient fractions. Even though we provide an example with heart mitochondria, this protocol can be directly applied to virtually all mouse tissues, as well as cultured cells, with little to no modifications.

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Authors and Affiliations

  1. Laboratory for Genetics of Mitochondrial Disorders, INSERM U1163, Université Paris Descartes-Sorbonne Paris Cité, Institut Imagine, Paris, France

    Benedetta Ruzzenente & Metodi D. Metodiev

Authors
  1. Benedetta Ruzzenente
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  2. Metodi D. Metodiev
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Corresponding author

Correspondence to Metodi D. Metodiev .

Editor information

Editors and Affiliations

  1. Basic Research Laboratory, National Cancer Institute, Frederick, MD, USA

    Shree Ram Singh

  2. Department of Surgery, University of California, AntiCancer, Inc., San Diego, CA, USA

    Robert M. Hoffman

  3. Department of Biology, University of Dayton, Dayton, OH, USA

    Amit Singh

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Ruzzenente, B., Metodiev, M.D. (2021). Linear Density Sucrose Gradients to Study Mitoribosomal Biogenesis in Tissue-Specific Knockout Mice. In: Singh, S.R., Hoffman, R.M., Singh, A. (eds) Mouse Genetics . Methods in Molecular Biology, vol 2224. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1008-4_3

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  • DOI: https://doi.org/10.1007/978-1-0716-1008-4_3

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1007-7

  • Online ISBN: 978-1-0716-1008-4

  • eBook Packages: Springer Protocols

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