Biochemistry

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    Protocols in Current Issue
    Expression and Purification of Yeast-derived GPCR, Gα and Gβγ Subunits for Structural and Dynamic Studies
    Authors:  Wenjie Zhao, Xudong Wang and Libin Ye, date: 02/20/2021, view: 940, Q&A: 0

    In the last several years, as evidence of a surged number of GPCR-G complex structures, the expressions of GPCRs and G proteins for structural biology have achieved tremendous successes, mostly in insect and mammalian cell systems, resulting in more

    ...
    Bioorthogonal Labeling and Chemoselective Functionalization of Lung Extracellular Matrix
    Authors:  Zihan Ling, Yunhui Xing, Emily Reinoso Jacome, Shierly W. Fok and Xi Ren, date: 02/20/2021, view: 607, Q&A: 0

    Decellularized extracellular matrix (ECM) biomaterials derived from native tissues and organs are widely used for tissue engineering and wound repair. To boost their regenerative potential, ECM biomaterials can be functionalized via the

    ...
    Extraction and Quantification of Sphingolipids from Hemiptera Insects by Ultra-Performance Liquid Chromatography Coupled to Tandem Mass Spectrometry
    Authors:  Ni Wang, Xiaoxiao Shi, Chao Zhang, Wenwu Zhou and Zeng-Rong Zhu, date: 02/20/2021, view: 562, Q&A: 0

    Sphingolipids are major structural components of endomembranes and have also been described as an intracellular second messenger involved in various biological functions in all eukaryotes and a few prokaryotes. Ceramides (Cer), the central molecules

    ...
    Tracing Nitrogen Metabolism in Mouse Tissues with Gas Chromatography-Mass Spectrometry
    Authors:  Rong Xu, Yekai Wang and Jianhai Du, date: 02/20/2021, view: 626, Q&A: 0

    Nitrogen-containing metabolites including ammonia, amino acids, and nucleotides, are essential for cell metabolism, growth, and neural transmission. Nitrogen metabolism is tightly coordinated with carbon metabolism in the breakdown and biosynthesis

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    Live Cell FRET Analysis of the Conformational Changes of Human P-glycoprotein
    Authors:  Ryota Futamata, Noriyuki Kioka and Kazumitsu Ueda, date: 02/20/2021, view: 728, Q&A: 0

    The molecular mechanisms of P-glycoprotein (P-gp; also known as MDR1 or ABCB1) have been mainly investigated using artificial membranes such as lipid-detergent mixed micelles, artificial lipid bilayers, and membrane vesicles derived from cultured

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    Live Cell FRET Analysis of the Conformational Changes of Human P-glycoprotein
    Authors:  Ryota Futamata, Noriyuki Kioka and Kazumitsu Ueda, date: 02/20/2021, view: 728, Q&A: 0
    [Abstract]

    The molecular mechanisms of P-glycoprotein (P-gp; also known as MDR1 or ABCB1) have been mainly investigated using artificial membranes such as lipid-detergent mixed micelles, artificial lipid bilayers, and membrane vesicles derived from cultured cells. Although these in vitro experiments help illustrate details about the molecular mechanisms of

    ...
    Tracing Nitrogen Metabolism in Mouse Tissues with Gas Chromatography-Mass Spectrometry
    Authors:  Rong Xu, Yekai Wang and Jianhai Du, date: 02/20/2021, view: 626, Q&A: 0
    [Abstract]

    Nitrogen-containing metabolites including ammonia, amino acids, and nucleotides, are essential for cell metabolism, growth, and neural transmission. Nitrogen metabolism is tightly coordinated with carbon metabolism in the breakdown and biosynthesis of amino acids and nucleotides. Both nuclear magnetic resonance spectroscopy and mass spectrometry

    ...
    Extraction and Quantification of Sphingolipids from Hemiptera Insects by Ultra-Performance Liquid Chromatography Coupled to Tandem Mass Spectrometry
    Authors:  Ni Wang, Xiaoxiao Shi, Chao Zhang, Wenwu Zhou and Zeng-Rong Zhu, date: 02/20/2021, view: 562, Q&A: 0
    [Abstract]

    Sphingolipids are major structural components of endomembranes and have also been described as an intracellular second messenger involved in various biological functions in all eukaryotes and a few prokaryotes. Ceramides (Cer), the central molecules of sphingolipids, have been depicted in cell growth arrest, cell differentiation, and apoptosis.

    ...
    Bioorthogonal Labeling and Chemoselective Functionalization of Lung Extracellular Matrix
    Authors:  Zihan Ling, Yunhui Xing, Emily Reinoso Jacome, Shierly W. Fok and Xi Ren, date: 02/20/2021, view: 607, Q&A: 0
    [Abstract]

    Decellularized extracellular matrix (ECM) biomaterials derived from native tissues and organs are widely used for tissue engineering and wound repair. To boost their regenerative potential, ECM biomaterials can be functionalized via the immobilization of bioactive molecules. To enable ECM functionalization in a chemoselective manner, we have

    ...
    Expression and Purification of Yeast-derived GPCR, Gα and Gβγ Subunits for Structural and Dynamic Studies
    Authors:  Wenjie Zhao, Xudong Wang and Libin Ye, date: 02/20/2021, view: 940, Q&A: 0
    [Abstract]

    In the last several years, as evidence of a surged number of GPCR-G complex structures, the expressions of GPCRs and G proteins for structural biology have achieved tremendous successes, mostly in insect and mammalian cell systems, resulting in more than 370 structures of over 70 GPCRs have been resolved. However, the challenge remains,

    ...
    Ecdysone Quantification from Whole Body Samples of Drosophila melanogaster Larvae
    Authors:  Takashi Koyama and Christen K. Mirth, date: 02/05/2021, view: 211, Q&A: 0
    [Abstract]

    Steroid hormones strictly control the timing of sexual maturation and final body size both in vertebrates and invertebrates. In insects, the steroid hormone ecdysone controls the timing of the molts between larval instars as well as the transition to metamorphosis. Growth during the final instar accounts for over 80% of the increase in final mass

    ...
    Plant ARGONAUTE Protein Immunopurification for Pathogen Cross Kingdom Small RNA Analysis
    Authors:  Florian Dunker, Bernhard Lederer and Arne Weiberg, date: 02/05/2021, view: 352, Q&A: 0
    [Abstract]

    Over the last decade, it has been noticed that microbial pathogens and pests deliver small RNA (sRNA) effectors into their host plants to manipulate plant physiology and immunity for infection, known as cross kingdom RNA interference. In this process, fungal and oomycete parasite sRNAs hijack the plant ARGONAUTE (AGO)/RNA-induced silencing complex

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    Relative Quantification of NaV1.1 Protein in Mouse Brains Using a Meso Scale Discovery-Electrochemiluminescence (MSD-ECL) Method
    Authors:  Zhou Han, Anne Christiansen, Meena and Gene Liau, date: 02/05/2021, view: 223, Q&A: 0
    [Abstract]

    Densitometric analysis is often used to quantify NaV1.1 protein on immunoblots, although the sensitivity and dilution linearity of the method are usually poor. Here we present a protocol for quantification of NaV1.1 in mouse brain tissues using a Meso Scale Discovery-Electrochemiluminescence (MSD-ECL) method. MSD-ECL is based on ELISA

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    Resolving Structural Changes of Photoreceptors in Living Escherichia coli via In-cell Infrared Difference Spectroscopy
    Authors:  Lukas Goett-Zink, Jessica L. Klocke and Tilman Kottke, date: 02/05/2021, view: 386, Q&A: 0
    [Abstract]

    Several in-cell spectroscopic techniques have been developed recently to investigate the structure and mechanism of proteins in their native environment. Conditions in vivo differ dramatically from those selected for in vitro experiments. Accordingly, the cellular environment can affect the protein mechanism for example by molecular crowding or

    ...
    Purification of Cytosolic Phospholipase A2α C2-domain after Expression in Soluble Form in Escherichia coli
    [Abstract]

    Previous expression/purification strategies for cytosolic phospholipase A2α C2-domain in Escherichia coli have relied on refolded protein recovered from inclusion bodies and sometimes containing C-terminal Cys139Ala and Cys141Ser substitutions to eliminate potential refolding complications induced by Cys residues. The protocol presented

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