Biochemistry

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    Protocols in Current Issue
    Efficient and Rapid Analysis of Polysomes and Ribosomal Subunits in Cells and Tissues Using Ribo Mega-SEC

    Polysome profile analysis is a popular method for separating polysomes and ribosomal subunits and is typically achieved using a sucrose density gradient (SDG). This has remained the gold standard method since ribosomes were first discovered;

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    Purification of Mitochondrial Ribosomes with the Translocase Oxa1L from HEK Cells
    Authors:  Hanting Yang and Nirupa Desai, date: 08/05/2021, view: 32, Q&A: 0

    Mitochondrial ribosomes (mitoribosomes) perform protein synthesis inside mitochondria, the organelles responsible for energy conversion and adenosine triphosphate (ATP) production in eukaryotic cells. To investigate their functions and structures,

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    Purification of Recombinant Wild Type and Mutant Ryanodine Receptors Expressed in HEK293 Cells

    High quantities of purified ryanodine receptor (RyR), a large (2.26 MDa) intracellular homotetrameric membrane protein, can be obtained from heterologous expression in HEK293 cells and used for structure determination by cryo-EM. The advantage of

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    Ion Transport Activity Assay for Microbial Rhodopsin Expressed in Escherichia coli Cells
    Authors:  Masae Konno, Keiichi Inoue and Hideki Kandori, date: 08/05/2021, view: 31, Q&A: 0

    Microbial rhodopsins have diverse functions, including roles as light-driven ion pumps, light-gated ion channels, photosensors, and light-regulated enzymes. As the number of rhodopsin-like genes identified has increased in recent years, so has the

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    Ion Transport Activity Assay for Microbial Rhodopsin Expressed in Escherichia coli Cells
    Authors:  Masae Konno, Keiichi Inoue and Hideki Kandori, date: 08/05/2021, view: 31, Q&A: 0
    [Abstract]

    Microbial rhodopsins have diverse functions, including roles as light-driven ion pumps, light-gated ion channels, photosensors, and light-regulated enzymes. As the number of rhodopsin-like genes identified has increased in recent years, so has the requirement for rapid identification of their functions. The patch-clamp method is often used to

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    Purification of Recombinant Wild Type and Mutant Ryanodine Receptors Expressed in HEK293 Cells
    [Abstract]

    High quantities of purified ryanodine receptor (RyR), a large (2.26 MDa) intracellular homotetrameric membrane protein, can be obtained from heterologous expression in HEK293 cells and used for structure determination by cryo-EM. The advantage of using recombinant protein is that the variability due to post-translational modifications can be

    ...
    Purification of Mitochondrial Ribosomes with the Translocase Oxa1L from HEK Cells
    Authors:  Hanting Yang and Nirupa Desai, date: 08/05/2021, view: 32, Q&A: 0
    [Abstract]

    Mitochondrial ribosomes (mitoribosomes) perform protein synthesis inside mitochondria, the organelles responsible for energy conversion and adenosine triphosphate (ATP) production in eukaryotic cells. To investigate their functions and structures, large-scale purification of intact mitoribosomes from mitochondria-rich animal tissues or HEK cells

    ...
    Efficient and Rapid Analysis of Polysomes and Ribosomal Subunits in Cells and Tissues Using Ribo Mega-SEC
    [Abstract]

    Polysome profile analysis is a popular method for separating polysomes and ribosomal subunits and is typically achieved using a sucrose density gradient (SDG). This has remained the gold standard method since ribosomes were first discovered; however, this method is time-consuming and requires multiple steps from making the gradient and long

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    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: 829, 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

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    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: 389, 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

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    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: 1128, 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

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    A Gel-Based Assay for Probing Protein Translocation on dsDNA
    Authors:  Christiane Brugger and Alexandra M. Deaconescu, date: 07/20/2021, view: 1060, 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

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    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: 818, 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

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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: 895, 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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