Protocols in Current Issue
    Preparation of Bacterial Outer Membrane Vesicles for Characterisation of Periplasmic Proteins in Their Native Environment
    Authors:  Johannes Thoma and Björn M. Burmann, date: 12/20/2020, view: 684, Q&A: 0

    Bacterial outer membrane vesicles (OMVs) are naturally formed by budding from the outer membrane of Gram-negative bacteria. OMVs consist of a lipid bilayer identical in composition to the original outer membrane and contain periplasmic content within their lumen. Enriched with specific envelope proteins, OMVs make for an excellent native-like

    Expression and Purification of Recombinant Skd3 (Human ClpB) Protein and Tobacco Etch Virus (TEV) Protease from Escherichia coli
    Authors:  Ryan R. Cupo and James Shorter, date: 12/05/2020, view: 695, Q&A: 0

    Skd3 (encoded by human CLPB) is a mitochondrial AAA+ protein comprised of an N-terminal ankyrin-repeat domain and a C-terminal HCLR-clade nucleotide-binding domain. The function of Skd3 has long remained unknown due to challenges in purifying the protein to high quality and near homogeneity. Recently we described Skd3 as a human mitochondrial

    Combining Gel Retardation and Footprinting to Determine Protein-DNA Interactions of Specific and/or Less Stable Complexes
    Authors:  Meng-Lun Hsieh, Alice Boulanger, Leslie G. Knipling and Deborah M. Hinton, date: 12/05/2020, view: 468, Q&A: 0

    DNA footprinting is a classic technique to investigate protein-DNA interactions. However, traditional footprinting protocols can be unsuccessful or difficult to interpret if the binding of the protein to the DNA is weak, the protein has a fast off-rate, or if several different protein-DNA complexes are formed. Our protocol differs from traditional

    In vitro Glutamylation Inhibition of Ubiquitin Modification and Phosphoribosyl-Ubiquitin Ligation Mediated by Legionella pneumophila Effectors
    Authors:  Alan G. Sulpizio, Marena E. Minelli and Yuxin Mao, date: 11/05/2020, view: 387, Q&A: 0

    Glutamylation is a posttranslational modification where the amino group of a free glutamate amino acid is conjugated to the carboxyl group of a glutamate side chain within a target protein. SidJ is a Legionella kinase-like protein that has recently been identified to perform protein polyglutamylation of the Legionella SdeA Phosphoribosyl-Ubiquitin

    Radioactive Assay of in vitro Glutamylation Activity of the Legionella pneumophila Effector Protein SidJ
    Authors:  Alan G. Sulpizio, Jung-Ho Shin, Marena E. Minelli and Yuxin Mao, date: 10/05/2020, view: 699, Q&A: 0
    [Abstract] The Legionella effector protein SidJ has recently been identified to perform polyglutamylation on another Legionella effector, SdeA, ablating SdeA’s activity. SidJ is a kinase-like protein that requires the small eukaryotic protein calmodulin to perform glutamylation. Glutamylation is a relatively uncommon type of ...
    A SsrA/NIa-based Strategy for Post-Translational Regulation of Protein Levels in Gram-negative Bacteria
    Authors:  Gonzalo Durante-Rodríguez, Belén Calles, Víctor de Lorenzo and Pablo I. Nikel, date: 07/20/2020, view: 866, Q&A: 0
    [Abstract] Strategies to control the levels of key enzymes of bacterial metabolism are commonly based on the manipulation of gene of interest within the target pathway. The development of new protocols towards the manipulation of biochemical processes is still a major challenge in the field of metabolic engineering. On this background, the FENIX (functional ...
    Identification of Buffer Conditions for Optimal Thermostability and Solubility of Herpesviral Protein UL37 Using the Thermofluor Assay
    Authors:  Andrea L. Koenigsberg, Jared D. Pitts and Ekaterina E. Heldwein, date: 06/20/2020, view: 954, Q&A: 0
    [Abstract] Structural and biochemical studies of proteins require high amounts of stable, purified proteins. Protein stability often depends on the buffer composition, which includes pH and concentration of salts or other solutes such as glycerol, hence an efficient method for identifying optimal buffer conditions for stability would minimize time and ...
    Differential Fractionation of Erythrocytes Infected by Plasmodium berghei
    Authors:  Bénédicte Gnangnon, Véronique Peucelle and Christine Pierrot, date: 06/05/2020, view: 980, Q&A: 0
    [Abstract] The study of host/pathogen interactions at the cellular level during Plasmodium intra-erythrocytic cycle requires differential extraction techniques aiming to analyze the different compartments of the infected cell. Various protocols have been proposed in the literature to study specific compartments and/or membranes in the infected ...
    A Sensitive Coupled Enzyme Assay for Measuring Kinase and ATPase Kinetics Using ADP-Specific Hexokinase
    Authors:  Ciaran R. McFarlane and James W. Murray, date: 05/05/2020, view: 1743, Q&A: 0
    [Abstract] Kinases and ATPases perform essential biological functions in metabolism and regulation. Activity of these enzymes is commonly measured by coupling ATP consumption to the synthesis of a detectable product. For most assay systems the ATP concentration during the reaction is unknown, compromising the precision of the assay.

    Using the ...
    Assessment of Metacaspase Activity in Phytoplankton
    Authors:  Dina Spungin and Ilana Berman-Frank, date: 08/20/2019, view: 2051, Q&A: 0
    [Abstract] Programmed cell death (PCD) is an irreversible, genetically-controlled form of cell suicide in which an endogenous biochemical pathway leads to morphological changes and ultimately, cellular demise. PCD is accompanied by de-novo protein synthesis of a family of proteases-"caspases" that are often used as a diagnostic marker of PCD. ...

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