Protocols in Current Issue
    Kinetic Characterization of the Shigella Type Three Secretion System ATPase Spa47 Using α-32P ATP
    Authors:  Heather B. Case and Nicholas E. Dickenson, date: 11/05/2018, view: 993, Q&A: 0
    [Abstract] ATPases represent a diverse class of enzymes that utilize ATP hydrolysis to support critical biological functions such as driving ion pumps, providing mechanical work, unfolding/folding proteins, and supporting otherwise thermodynamically unfavorable chemical reactions. We have recently shown that the Shigella protein Spa47 is an ATPase ...
    Detection of Catalase Activity by Polyacrylamide Gel Electrophoresis (PAGE) in Cell Extracts from Pseudomonas aeruginosa
    Authors:  Magdalena Pezzoni, Ramón A. Pizarro and Cristina S. Costa, date: 06/05/2018, view: 2092, Q&A: 0
    [Abstract] Bacteria in nature and as pathogens commonly face oxidative stress which causes damage to proteins, lipids and DNA. This damage is produced by the action of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), singlet oxygen, superoxide anion and hydroxyl radical. ROS are generated by antimicrobials, environmental ...
    In vitro Analysis of Ubiquitin-like Protein Modification in Archaea
    Authors:  Xian Fu, Zachary Adams and Julie A. Maupin-Furlow, date: 05/20/2018, view: 1817, Q&A: 0
    [Abstract] The ubiquitin-like (Ubl) protein is widely distributed in Archaea and involved in many cellular pathways. A well-established method to reconstitute archaeal Ubl protein conjugation in vitro is important to better understand the process of archaeal Ubl protein modification. This protocol describes the in vitro reconstitution of ...
    Heterologous Expression and Purification of the CRISPR-Cas12a/Cpf1 Protein
    Authors:  Prarthana Mohanraju, John van der Oost, Martin Jinek and Daan C. Swarts, date: 05/05/2018, view: 3940, Q&A: 3
    [Abstract] This protocol provides step by step instructions (Figure 1) for heterologous expression of Francisella novicida Cas12a (previously known as Cpf1) in Escherichia coli. It additionally includes a protocol for high-purity purification and briefly describes how activity assays can be performed. These protocols can also be used for ...
    Superoxide Dismutase (SOD) and Catalase (CAT) Activity Assay Protocols for Caenorhabditis elegans
    Authors:  Jing Zhang, Rui Chen, Zhenyang Yu and Lili Xue, date: 08/20/2017, view: 14033, Q&A: 0
    [Abstract] Assays for superoxide dismutase (SOD) and catalase (CAT) activities are widely employed to indicate antioxidant responses underlying the toxic effects of test chemicals. Yet, earlier studies mainly described the procedures as performed according to manufacturer’s instructions without modifications that are specific to any organisms. The present ...
    Liposome Disruption Assay to Examine Lytic Properties of Biomolecules
    Authors:  John R. Jimah, Paul H. Schlesinger and Niraj H. Tolia, date: 08/05/2017, view: 5511, Q&A: 0
    [Abstract] Proteins may have three dimensional structural or amino acid features that suggest a role in targeting and disrupting lipids within cell membranes. It is often necessary to experimentally investigate if these proteins and biomolecules are able to disrupt membranes in order to conclusively characterize the function of these biomolecules. Here, we ...
    Measurement of Energy-dependent Rhodamine 6G Efflux in Yeast Species
    Authors:  Yvetta Gbelska, Nora Toth Hervay, Vladimira Dzugasova and Alexandra Konecna, date: 08/05/2017, view: 3671, Q&A: 0
    [Abstract] Rhodamine 6G is a highly fluorescent dye often used to determine the transport activity of yeast membrane efflux pumps. The ATP-binding cassette transporter KlPdr5p confers resistance to several unrelated drugs in Kluyveromyces lactis. KlPdr5p also extrudes rhodamine 6G (R6G) from intact yeast cells in an ...
    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: 3088, 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: 3657, 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 ...
    Expression and Purification of Cyanobacterial Circadian Clock Protein KaiC and Determination of Its Auto-phosphatase Activity
    Authors:  Qiang Chen, Lingling Yu, Xiao Tan and Sen Liu, date: 02/20/2017, view: 3975, Q&A: 0
    [Abstract] Circadian rhythms are biological processes displaying an endogenous oscillation with a period of ~24 h. They allow organisms to anticipate and get prepared for the environmental changes caused mainly by the rotation of Earth. Circadian rhythms are driven by circadian clocks that consist of proteins, DNA, and/or RNA. Circadian clocks of ...

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