Biochemistry

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    Protocols in Current Issue
    Proteomics Profiling of S-sulfurated Proteins in Acinetobacter baumannii
    Authors:  Brenna J. C. Walsh and David P. Giedroc, date: 05/05/2021, view: 243, Q&A: 0

    Hydrogen sulfide (H2S) is emerging as an important modulator in bacterial cytoprotection against the host immune response in infected animals, which may well be attributed to downstream highly oxidized sulfur species, termed reactive sulfur species

    ...
    Click Chemistry for Imaging in-situ Protein Palmitoylation during the Asexual Stages of Plasmodium falciparum
    Author:  Mansoor A. Siddiqui, date: 05/05/2021, view: 385, Q&A: 0

    Palmitoylation refers to the modification of the cysteine thiols in proteins by fatty acids, most commonly palmitic acid, through ‘thioester bond’ formation. In vivo, palmitoylation of proteins is catalyzed by palmitoyl acyltransferases

    ...
    Spin Labeling of RNA Using “Click” Chemistry for Coarse-grained Structure Determination via Pulsed Electron-electron Double Resonance Spectroscopy
    Authors:  Maria F. Vicino, Tobias Hett and Olav Schiemann, date: 05/05/2021, view: 222, Q&A: 0

    Understanding the function of oligonucleotides on a molecular level requires methods for studying their structure, conformational changes, and internal dynamics. Various biophysical methods exist to achieve this, including the whole toolbox of

    ...
    COVID-19 Sample Pooling: From RNA Extraction to Quantitative Real-time RT-PCR

    The COVID-19 pandemic requires mass screening to identify those infected for isolation and quarantine. Individually screening large populations for the novel pathogen, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is costly and

    ...
    Histological Methods to Detect Early-stage Plant Defense Responses during Artificial Inoculation of Lolium perenne with Epichloë festucae
    Authors:  Mostafa Rahnama, Damien J. Fleetwood and Richard D. Johnson, date: 05/05/2021, view: 180, Q&A: 0

    Epichloë species form agriculturally important symbioses with many cool season grasses. To study these symbioses, such as the interaction of Epichloë festucae with perennial ryegrass (Lolium perenne), host plants can be infected by

    ...
    Chromatographic Assays for the Enzymatic Degradation of Chitin
    Authors:  Sophanit Mekasha, Tina R. Tuveng, Gustav Vaaje-Kolstad and Vincent G. H. Eijsink, date: 05/05/2021, view: 307, Q&A: 0

    Chitin is an insoluble linear polymer of β(1→4)-linked N-acetylglucosamine. Enzymatic cleavage of chitin chains can be achieved using hydrolytic enzymes, called chitinases, and/or oxidative enzymes, called lytic polysaccharide

    ...
    Characterising Plant Deubiquitinases with in vitro Activity-based Labelling and Ubiquitin Chain Disassembly Assays
    Authors:  Michael J. Skelly and Steven H. Spoel, date: 05/05/2021, view: 242, Q&A: 0

    Post-translational modification of proteins by ubiquitin is an essential cellular signaling mechanism in all eukaryotes. Ubiquitin is removed from target proteins by a wide range of deubiquitinase (DUB) enzymes with different activities and

    ...
    Characterising Plant Deubiquitinases with in vitro Activity-based Labelling and Ubiquitin Chain Disassembly Assays
    Authors:  Michael J. Skelly and Steven H. Spoel, date: 05/05/2021, view: 242, Q&A: 0
    [Abstract]

    Post-translational modification of proteins by ubiquitin is an essential cellular signaling mechanism in all eukaryotes. Ubiquitin is removed from target proteins by a wide range of deubiquitinase (DUB) enzymes with different activities and substrate specificities. Understanding how DUBs function in vitro is a vital first step to uncovering their

    ...
    Chromatographic Assays for the Enzymatic Degradation of Chitin
    Authors:  Sophanit Mekasha, Tina R. Tuveng, Gustav Vaaje-Kolstad and Vincent G. H. Eijsink, date: 05/05/2021, view: 307, Q&A: 0
    [Abstract]

    Chitin is an insoluble linear polymer of β(1→4)-linked N-acetylglucosamine. Enzymatic cleavage of chitin chains can be achieved using hydrolytic enzymes, called chitinases, and/or oxidative enzymes, called lytic polysaccharide monooxygenases (LPMOs). These two groups of enzymes have different modes of action and yield different product

    ...
    Histological Methods to Detect Early-stage Plant Defense Responses during Artificial Inoculation of Lolium perenne with Epichloë festucae
    Authors:  Mostafa Rahnama, Damien J. Fleetwood and Richard D. Johnson, date: 05/05/2021, view: 180, Q&A: 0
    [Abstract]

    Epichloë species form agriculturally important symbioses with many cool season grasses. To study these symbioses, such as the interaction of Epichloë festucae with perennial ryegrass (Lolium perenne), host plants can be infected by artificial inoculation of etiolated seedlings. This inoculation is performed by placing mycelium into an

    ...
    COVID-19 Sample Pooling: From RNA Extraction to Quantitative Real-time RT-PCR
    [Abstract]

    The COVID-19 pandemic requires mass screening to identify those infected for isolation and quarantine. Individually screening large populations for the novel pathogen, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is costly and requires a lot of resources. Sample pooling methods improve the efficiency of mass screening and consume

    ...
    Spin Labeling of RNA Using “Click” Chemistry for Coarse-grained Structure Determination via Pulsed Electron-electron Double Resonance Spectroscopy
    Authors:  Maria F. Vicino, Tobias Hett and Olav Schiemann, date: 05/05/2021, view: 222, Q&A: 0
    [Abstract]

    Understanding the function of oligonucleotides on a molecular level requires methods for studying their structure, conformational changes, and internal dynamics. Various biophysical methods exist to achieve this, including the whole toolbox of Electron Paramagnetic Resonance (EPR or ESR) spectroscopy. An EPR method widely used in this regard is

    ...
    Click Chemistry for Imaging in-situ Protein Palmitoylation during the Asexual Stages of Plasmodium falciparum
    Author:  Mansoor A. Siddiqui, date: 05/05/2021, view: 385, Q&A: 0
    [Abstract]

    Palmitoylation refers to the modification of the cysteine thiols in proteins by fatty acids, most commonly palmitic acid, through ‘thioester bond’ formation. In vivo, palmitoylation of proteins is catalyzed by palmitoyl acyltransferases (PATs or DHHC-PATs). Palmitoylation has recently emerged as a crucial post-translational

    ...
    Proteomics Profiling of S-sulfurated Proteins in Acinetobacter baumannii
    Authors:  Brenna J. C. Walsh and David P. Giedroc, date: 05/05/2021, view: 243, Q&A: 0
    [Abstract]

    Hydrogen sulfide (H2S) is emerging as an important modulator in bacterial cytoprotection against the host immune response in infected animals, which may well be attributed to downstream highly oxidized sulfur species, termed reactive sulfur species (RSS), derived from H2S. One mechanism by which H2S/RSS may signal in the cell is through proteome

    ...
    Optimized Recombinant Production of Secreted Proteins Using Human Embryonic Kidney (HEK293) Cells Grown in Suspension
    [Abstract]

    Recombinant proteins are an essential milestone for a plethora of different applications ranging from pharmaceutical to clinical, and mammalian cell lines are among the currently preferred systems to obtain large amounts of proteins of interest due to their high level of post-translational modification and manageable large-scale production. In

    ...
    Quantitative Characterization of the Amount and Length of (1,3)-β-D-glucan for Functional and Mechanistic Analysis of Fungal (1,3)-β-D-glucan Synthase
    Authors:  Abhishek Chhetri, Anna Loksztejn and Kenichi Yokoyama, date: 04/20/2021, view: 818, Q&A: 0
    [Abstract]

    (1,3)-β-d-Glucan synthase (GS) is an essential enzyme for fungal cell wall biosynthesis that catalyzes the synthesis of (1,3)-β-d-glucan, a major and vital component of the cell wall. GS is a proven target of antifungal antibiotics including FDA-approved echinocandin derivatives; however, the function and mechanism of GS remain largely

    ...
    Reconstitution of Chromatin by Stepwise Salt Dialysis
    Authors:  Grisel Cruz-Becerra and James T. Kadonaga, date: 04/05/2021, view: 338, Q&A: 0
    [Abstract]

    Chromatin, rather than plain DNA, is the natural substrate of the molecular machines that mediate DNA-directed processes in the nucleus. Chromatin can be reconstituted in vitro by using different methodologies. The salt dialysis method yields chromatin that consists of purified histones and DNA. This biochemically pure chromatin is well-suited for

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