Prokaryotes

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    Protocols in Current Issue
    A Method for User-defined Mutagenesis by Integrating Oligo Pool Synthesis Technology with Nicking Mutagenesis
    Authors:  Paul J. Steiner, Zachary T Baumer and Timothy A. Whitehead, date: 08/05/2020, view: 791, Q&A: 0
    [Abstract] Saturation mutagenesis is a fundamental enabling technology for protein engineering and epitope mapping. Nicking mutagenesis (NM) allows the user to rapidly construct libraries of all possible single mutations in a target protein sequence from plasmid DNA in a one-pot procedure. Briefly, one strand of the plasmid DNA is degraded using a nicking ...
    Sequence Alignment Using Machine Learning for Accurate Template-based Protein Structure Prediction
    Authors:  Shuichiro Makigaki and Takashi Ishida, date: 05/05/2020, view: 1080, Q&A: 0
    [Abstract] Template-based modeling, the process of predicting the tertiary structure of a protein by using homologous protein structures, is useful when good templates can be available. Indeed, modern homology detection methods can find remote homologs with high sensitivity. However, the accuracy of template-based models generated from the ...
    Preparation, FPLC Purification and LC-FT-ICR-MS of Proteins
    Authors:  Tyler B. Johnson, Jiri Adamec and Paul Blum, date: 04/05/2020, view: 1128, Q&A: 0
    [Abstract] High magnetic field Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers provide extremely high mass resolution (resolving power of ~200,000 at 400 m/z) protein detection across a broad mass range, enabling analysis of fine structure of isotopic peak clusters that is missed in other types of mass spectrometers. The protocol ...
    The Sulfur Oxygenase Reductase Activity Assay: Catalyzing a Reaction with Elemental Sulfur as Substrate at High Temperatures
    Authors:  Patrick Rühl and Arnulf Kletzin, date: 07/20/2017, view: 4676, Q&A: 0
    [Abstract] The sulfur oxygenase reductase (SOR) reaction is a dioxygen-dependent disproportionation of elemental sulfur (S0), catalyzed at optimal temperatures between 65 °C and 85 °C. Thiosulfate and sulfite are formed as oxidized products as well hydrogen sulfide as reduced product. External co-factors are not required. Usually, the SOR assay is ...
    Assays for the Detection of Rubber Oxygenase Activities
    Authors:  Wolf Röther, Jakob Birke and Dieter Jendrossek, date: 03/20/2017, view: 5118, Q&A: 0
    [Abstract] Microbial biodegradation of rubber relies on extracellular rubber oxygenases that catalyze the oxidative cleavage of the double bond of the polyisoprene backbone into oligo-isoprenoids. This protocol describes the determination of rubber oxygenase activities by an online measurement of molecular oxygen consumption via a non-invasive ...
    Semi-denaturing Detergent Agarose Gel Electrophoresis (SDD-AGE)
    Authors:  Laura Molina-García and Fátima Gasset-Rosa, date: 11/20/2014, view: 15040, Q&A: 2
    [Abstract] Pathological proteins in neurodegenerative diseases suffer a conformational change to a misfolded amyloid state. Such pathological event leads to the aggregation of these proteins that indefinitely propagates as an altered form of itself, and harbor prion-like properties (Wickner, 1994; Prusiner, 2012). In addition to diseases, prions can also ...
    Purification and Detection of a PDGA Depolymerase from Pusillimonas noertemannii
    Authors:  David Negus and Peter W. Taylor, date: 11/05/2014, view: 6741, Q&A: 0
    [Abstract] The purification of a target protein from a complex mixture of proteins is a challenging undertaking. If the target protein has been previously characterised, then information such as subcellular location, function, molecular weight and pI can be used for the design of a purification strategy. However, if the target protein is uncharacterised or ...
    Accelerated Storage Stability Testing of a Potential Anti-Anthrax Therapeutic, EnvD
    Authors:  David Negus and Peter W. Taylor, date: 11/05/2014, view: 6242, Q&A: 0
    [Abstract] The purpose of stability testing is to determine how the properties of a particular therapeutic vary with time under the influence of specific environmental factors. Information regarding the long-term stability of therapeutics can be extrapolated by performing an accelerated storage stability study. Here, we describe an accelerated storage ...
    Fractionation by Ultracentrifugation of Gram Negative Cytoplasmic and Membrane Proteins
    Authors:  Sara M Sandrini, Richard Haigh and Primrose P. E. Freestone, date: 11/05/2014, view: 15820, Q&A: 1
    [Abstract] Protein fractionation is a useful separation process which divides membrane proteins (including those located in the outer and inner membrane) and cytoplasmic proteins into discrete fractions. Fractionation of proteins can simplify analysis of the numbers of proteins present, and therefore make easier to characterize any environmentally or ...
    In vitro Assay for Cytidine Deaminase Activity of APOBEC3 Protein
    Authors:  Smita Nair and Alan Rein, date: 10/20/2014, view: 9778, Q&A: 1
    [Abstract] Cytidine deaminases are enzymes that catalyze the removal of an amino group from cytidine, forming uridine. APOBEC3 (ApolipoproteinB mRNA editing enzyme, catalytic polypeptide like) proteins are cytidine deaminases that deaminate cytidines in polynucleotides (RNA/DNA), resulting in editing of their target substrates. Mammalian APOBEC3 proteins are ...



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