Protocols in Current Issue
    Analysis of Protein Stability by Synthesis Shutoff
    Authors:  Ira Buntenbroich, Tânia Simões and Mafalda Escobar-Henriques, date: 11/20/2021, view: 1216, Q&A: 0

    In this protocol, we describe the analysis of protein stability over time, using synthesis shutoff. As an example, we express HA-tagged yeast mitofusin Fzo1 in Saccharomyces cerevisiae and inhibit translation via cycloheximide (CHX). Proteasomal inhibition with MG132 is performed, as an optional step, before the addition of CHX. Proteins are

    Isolation and Phospholipid Enrichment of Muscle Mitochondria and Mitoplasts

    The efficient ATP production in mitochondria relies on the highly specific organization of its double membrane. Notably, the inner mitochondrial membrane (IMM) displays a massive surface extension through its folding into cristae, along which concentrate respiratory complexes and oligomers of the ATP synthase. Evidence has accumulated to highlight

    An ex vivo Approach to Assess Mitochondrial ROS by Flow Cytometry in AAV-tagged Astrocytes in Adult Mice
    Authors:  Carlos Vicente-Gutierrez and Juan P. Bolaños, date: 03/20/2020, view: 3650, Q&A: 0
    [Abstract] Mitochondrial reactive oxygen species (mROS) are naturally produced signalling molecules extremely relevant for understanding both health- and disease-associated biological processes. The study of mROS in the brain is currently underway to decipher their physiopathological roles and contributions in neurological diseases. Recent advances in this ...
    Analysis of the Mitochondrial Membrane Potential Using the Cationic JC-1 Dye as a Sensitive Fluorescent Probe
    Authors:  Farzane Sivandzade, Aditya Bhalerao and Luca Cucullo, date: 01/05/2019, view: 22052, Q&A: 0
    [Abstract] In recent years, fluorescent dyes have been frequently used for monitoring mitochondrial membrane potential to evaluate mitochondrial viability and function. However, the reproducibility of the results across laboratories strongly depends upon following well validated and reliable protocols along with the appropriate controls. Herein, we provide a ...
    Image-Based Analysis of Mitochondrial Area and Counting from Adult Mouse Dopaminergic Neurites
    Authors:  Nadee Nissanka, Carlos T Moraes and Mlena Pinto, date: 08/20/2018, view: 7103, Q&A: 0
    [Abstract] Mitochondria form dynamic cytoplasmic networks which undergo morphological changes in order to adapt to cellular stresses and signals. These changes can include alterations in size and number within a given cell. Analysis of the whole network can be a useful metric to assess overall mitochondrial health, particularly in neurons, which are highly ...
    Non-radioactive in vitro PINK1 Kinase Assays Using Ubiquitin or Parkin as Substrate
    Authors:  Fabienne C. Fiesel, Roman Hudec and Wolfdieter Springer, date: 10/05/2016, view: 7262, Q&A: 0
    [Abstract] This protocol describes the in vitro phosphorylation of ubiquitin and Parkin by the kinase PINK1 using recombinant proteins. Both substrates, ubiquitin and Parkin, are phosphorylated at the conserved serine 65 residue (pS65-ubiquitin and pS65-Parkin). The protocol also includes the use of monomeric and K48- and K63-linked poly-ubiquitin ...
    Isolating Brain Mitochondria by Differential Centrifugation
    Authors:  Ignacio Amigo, Javier Traba and Carlos B. Rueda, date: 05/20/2016, view: 15099, Q&A: 3
    [Abstract] In addition to methods aimed at the study of mitochondrial function in-situ, a full understanding of mitochondrial function requires their purification from cells or tissues under specific physiological or pathological conditions. This protocol illustrates a sequential procedure to obtain functional mitochondria with high yield from mice ...
    Mitochondrial Isolation and Purification from Mouse Spinal Cord
    Authors:  Philippe A. Parone, Sandrine Da Cruz and Don W. Cleveland, date: 11/05/2013, view: 10695, Q&A: 0
    [Abstract] Mitochondria are eukaryotic organelles that play a crucial role in several cellular processes, including energy production, β-oxidation of fatty acids and regulation of calcium homeostasis. In the last 20 years there has been a hightened interest in the study of mitochondria following the discoveries that mitochondria are central to the process of ...

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