Protocols in Current Issue
    Analysis of Direct Interaction between Viral DNA-binding Proteins by Protein Pull-down Co-immunoprecipitation Assay
    Authors:  Ana Lechuga, Mónica Berjón-Otero, Margarita Salas and Modesto Redrejo-Rodríguez, date: 01/05/2018, view: 435, Q&A: 0
    [Abstract] This protocol analyzes the direct interaction between two DNA-binding proteins by pull-down co-immunoprecipitation. One of the proteins is overexpressed in E. coli as HA-tagged recombinant protein and cell-free extracts are immunoprecipitated in HA-affinity resin. Cell extracts are treated with nuclease to degrade DNA and RNA, which rules ...
    Easy and Efficient Permeabilization of Cyanobacteria for in vivo Enzyme Assays Using B-PER
    Authors:  Simon Matthé Erstad and Yumiko Sakuragi, date: 01/05/2018, view: 293, Q&A: 0
    [Abstract] Cyanobacteria are photosynthetic bacteria that thrive in diverse ecosystems and play major roles in the global carbon cycle. The abilities of cyanobacteria to fix atmospheric CO2 and to allocate the fixed carbons to chemicals and biofuels have attracted growing attentions as sustainable microbial cell factories. A better understanding ...
    Snapshots of the Signaling Complex DesK:DesR in Different Functional States Using Rational Mutagenesis and X-ray Crystallography
    [Abstract] We have developed protocols to generate site-specific variants of the histidine-kinase DesK and its cognate response regulator DesR, conducive to trapping different signaling states of the proteins. Co-expression of both partners in E. coli, ensuring an excess of the regulator, was essential for soluble production of the DesK:DesR ...
    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: 1739, 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: 1779, 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: 1175, 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 ...
    Mapping RNA Sequences that Contact Viral Capsid Proteins in Virions
    Authors:  C. Cheng Kao, Ella Chuang, James Ford, Jie Huang, Ram Podicheti and Doug B. Rusch, date: 07/20/2017, view: 1278, Q&A: 0
    [Abstract] We have adapted the methodology of CLIP-seq (Crosslinking-Immunoprecipitation and DNA Sequencing) to map the segments of encapsidated RNAs that contact the protein shells of virions. Results from the protocol report on the RNA sequences that contact the viral capsid.
    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: 1065, 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 ...
    Producing GST-Cbx7 Fusion Proteins from Escherichia coli
    Authors:  Thao Ngoc Huynh and Xiaojun Ren , date: 06/20/2017, view: 1430, Q&A: 1
    [Abstract] This protocol describes the production of GST-Cbx7 fusion proteins from E. coli, originally developed in the recent publication (Zhen et al., 2016). The pGEX-6P-1-GST plasmids encoding the Cbx7 variants were transformed into BL21 competent cells. The fusion protein production was induced by isopropyl-beta-D-thiogalactopyranoside ...
    Expression and Purification of the Cas10-Csm Complex from Staphylococci
    Authors:  Lucy Chou-Zheng and Asma Hatoum-Aslan, date: 06/05/2017, view: 2044, Q&A: 0
    [Abstract] CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) is a class of prokaryotic immune systems that degrade foreign nucleic acids in a sequence-specific manner. These systems rely upon ribonucleoprotein complexes composed of Cas nucleases and small CRISPR RNAs (crRNAs). Staphylococcus epidermidis ...