Biochemistry

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    Protocols in Current Issue
    Analysis of the Effects of Hexokinase 2 Detachment From Mitochondria-Associated Membranes with the Highly Selective Peptide HK2pep
    Authors:  Francesco Ciscato, Federica Chiara, Riccardo Filadi and Andrea Rasola, date: 07/20/2021, view: 788, Q&A: 0

    The crucial role of hexokinase 2 (HK2) in the metabolic rewiring of tumors is now well established, which makes it a suitable target for the design of novel therapies. However, hexokinase activity is central to glucose utilization in all tissues;

    ...
    A Gel-Based Assay for Probing Protein Translocation on dsDNA
    Authors:  Christiane Brugger and Alexandra M. Deaconescu, date: 07/20/2021, view: 1006, Q&A: 0

    Protein translocation on DNA represents the key biochemical activity of ssDNA translocases (aka helicases) and dsDNA translocases such as chromatin remodelers. Translocation depends on DNA binding but is a distinct process as it typically involves

    ...
    Modeling Perturbations in Protein Filaments at the Micro and Meso Scale Using NAMD and PTools/Heligeom
    Authors:  Benjamin Boyer, Benoist Laurent, Charles H. Robert and Chantal Prévost, date: 07/20/2021, view: 1018, Q&A: 0

    Protein filaments are dynamic entities that respond to external stimuli by slightly or substantially modifying the internal binding geometries between successive protomers. This results in overall changes in the filament architecture, which are

    ...
    In vitro Nitrate Reductase Activity Assay of Mycolicibacterium smegmatis Crude Extract
    Authors:  Wei Tan, Zhi-Hui Shao and Guo-Ping Zhao, date: 07/20/2021, view: 358, Q&A: 0

    Nitrate is one of the major inorganic nitrogen sources for microorganisms. Many bacterial and archaeal lineages can express cytoplasmic assimilatory nitrate reductase (NAS), which catalyzes the rate-limiting reduction of nitrate to nitrite in the

    ...
    Protocol for Spontaneous and Chaperonin-assisted in vitro Refolding of a Slow-folding Mutant of GFP, sGFP
    Authors:  Anwar Sadat, Satyam Tiwari and Koyeli Mapa, date: 07/20/2021, view: 798, Q&A: 0

    Understanding the folding pathway of any protein is of utmost importance for deciphering the folding problems under adverse conditions. We can obtain important information about the folding pathway by monitoring the folding of any protein from its

    ...
    Analysis of the Effects of Hexokinase 2 Detachment From Mitochondria-Associated Membranes with the Highly Selective Peptide HK2pep
    Authors:  Francesco Ciscato, Federica Chiara, Riccardo Filadi and Andrea Rasola, date: 07/20/2021, view: 788, Q&A: 0
    [Abstract]

    The crucial role of hexokinase 2 (HK2) in the metabolic rewiring of tumors is now well established, which makes it a suitable target for the design of novel therapies. However, hexokinase activity is central to glucose utilization in all tissues; thus, enzymatic inhibition of HK2 can induce severe adverse effects. In an effort to find a selective

    ...
    A Gel-Based Assay for Probing Protein Translocation on dsDNA
    Authors:  Christiane Brugger and Alexandra M. Deaconescu, date: 07/20/2021, view: 1006, Q&A: 0
    [Abstract]

    Protein translocation on DNA represents the key biochemical activity of ssDNA translocases (aka helicases) and dsDNA translocases such as chromatin remodelers. Translocation depends on DNA binding but is a distinct process as it typically involves multiple DNA binding states, which are usually dependent on nucleotide binding/hydrolysis and are

    ...
    Modeling Perturbations in Protein Filaments at the Micro and Meso Scale Using NAMD and PTools/Heligeom
    Authors:  Benjamin Boyer, Benoist Laurent, Charles H. Robert and Chantal Prévost, date: 07/20/2021, view: 1018, Q&A: 0
    [Abstract]

    Protein filaments are dynamic entities that respond to external stimuli by slightly or substantially modifying the internal binding geometries between successive protomers. This results in overall changes in the filament architecture, which are difficult to model due to the helical character of the system. Here, we describe how distortions in RecA

    ...
    In vitro Nitrate Reductase Activity Assay of Mycolicibacterium smegmatis Crude Extract
    Authors:  Wei Tan, Zhi-Hui Shao and Guo-Ping Zhao, date: 07/20/2021, view: 358, Q&A: 0
    [Abstract]

    Nitrate is one of the major inorganic nitrogen sources for microorganisms. Many bacterial and archaeal lineages can express cytoplasmic assimilatory nitrate reductase (NAS), which catalyzes the rate-limiting reduction of nitrate to nitrite in the nitrate assimilation pathway. Here, we present a detailed protocol for measuring in vitro nitrate

    ...
    Protocol for Spontaneous and Chaperonin-assisted in vitro Refolding of a Slow-folding Mutant of GFP, sGFP
    Authors:  Anwar Sadat, Satyam Tiwari and Koyeli Mapa, date: 07/20/2021, view: 798, Q&A: 0
    [Abstract]

    Understanding the folding pathway of any protein is of utmost importance for deciphering the folding problems under adverse conditions. We can obtain important information about the folding pathway by monitoring the folding of any protein from its unfolded state. It is usually very difficult to monitor the folding process in real time as the

    ...
    Visualization of Host Cell Kinase Activation by Viral Proteins Using GFP Fluorescence Complementation and Immunofluorescence Microscopy
    Authors:  Sherry T. Shu, Wing Fai Li and Thomas E. Smithgall, date: 07/05/2021, view: 1426, Q&A: 0
    [Abstract]

    Non-receptor protein-tyrosine kinases regulate cellular responses to many external signals and are important drug discovery targets for cancer and infectious diseases. While many assays exist for the assessment of kinase activity in vitro, methods that report changes in tyrosine kinase activity in single cells have the potential to provide

    ...
    Monitoring Changes in the Oxidizing Milieu in the Endoplasmic Reticulum of Mammalian Cells Using HyPerER
    Authors:  Julia Birk, Beata Lizak, Christian Appenzeller-Herzog and Alex Odermatt, date: 07/05/2021, view: 1272, Q&A: 0
    [Abstract]

    The production of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress are tightly linked. The generation of ROS can be both the cause and a consequence of ER stress pathways, and an increasing number of human diseases are characterized by tissue atrophy in response to ER stress and oxidative injury. For the assessment of modulators

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    Analysis of Soluble Sugar Content in Minute Quantities of Rice Tissues by GC-MS
    Authors:  Li-he Xu, Li-ying Xiao, Xue-qiong Xiao, Yan-nong Xiao and Gao-feng Wang, date: 07/05/2021, view: 1259, Q&A: 0
    [Abstract]

    Soluble sugars play key roles in plant growth, development, and adaption to the environment. Characterizing sugar content profiling of plant tissues promotes our understanding of the mechanisms underlying these plant processes. Several technologies have been developed to quantitate soluble sugar content in plant tissues; however, it is difficult

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    Immunofluorescence of GFAP and TNF-α in the Mouse Hypothalamus
    Authors:  Prasad S. Dalvi and Denise D. Belsham, date: 07/05/2021, view: 667, Q&A: 0
    [Abstract]

    Immunofluorescence is a reliable method for identifying specific proteins in neuronal and glial cell populations of the hypothalamus. Several immunofluorescence protocols are available to detect protein markers and neuropeptides in the hypothalamus; however, published methods may vary in subtle details that can potentially impact the final outcome

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    Yeast Lipid Extraction and Analysis by HPTLC
    Authors:  Dan Li, Zheng-Tan Zhang, Cheng-Wen He and Zhiping Xie, date: 07/05/2021, view: 872, Q&A: 0
    [Abstract]

    The diversity of lipid structures, properties, and combinations in biological tissues makes their extraction and analysis an experimental challenge. Accordingly, even for one of the simplest single-cellular fungi, the budding yeast (Saccharomyces cerevisiae), numerous extraction and analysis protocols have been developed to separate and quantitate

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