Microbiology

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    Protocols in Current Issue
    Assessment of Diadenylate Cyclase and c-di-AMP-phosphodiesterase Activities Using Thin-layer and Ion Exchange Chromatography
    Authors:  Andreas Latoscha, David Jan Drexler, Gregor Witte and Natalia Tschowri, date: 01/05/2021, view: 331, Q&A: 0

    All living cells use cyclic nucleotides as second messengers for signal sensing and transduction. Cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) is primarily involved in the control of bacterial and euryarcheal osmoadaptation and is

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    Assessment of Diadenylate Cyclase and c-di-AMP-phosphodiesterase Activities Using Thin-layer and Ion Exchange Chromatography
    Authors:  Andreas Latoscha, David Jan Drexler, Gregor Witte and Natalia Tschowri, date: 01/05/2021, view: 331, Q&A: 0
    [Abstract]

    All living cells use cyclic nucleotides as second messengers for signal sensing and transduction. Cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) is primarily involved in the control of bacterial and euryarcheal osmoadaptation and is produced by diadenylate cyclases from two molecules of ATP. Specific phosphodiesterases hydrolyze

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    Evaluation of the Sequence Variability within the PCR Primer/Probe Target Regions of the SARS-CoV-2 Genome
    Authors:  Kashif Aziz Khan and Peter Cheung, date: 12/20/2020, view: 668, Q&A: 0
    [Abstract] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; initially named 2019-nCoV) is responsible for the recent coronavirus disease (COVID-19) pandemic, and polymerase chain reaction (PCR) is the current standard method for diagnosis from patient samples. As PCR assays are prone to sequence mismatches due to mutations in the viral genome, it ...
    Antimicrobial Sensitivity Assay for Bdellovibrio bacteriovorus
    Authors:  Emanuele Marine and Klaas M. Pos, date: 12/20/2020, view: 474, Q&A: 0
    [Abstract]

    Bdellovibrio bacteriovorus, an obligate predatory bacterium [i.e., bacteria that kill and feed on other bacteria (prey)], has the potential to be used as a probiotic for the disinfection of surfaces or for the treatment of bacterial infections. One option is to use this organism in combination with antimicrobials to potentiate the effectiveness of

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    Pea Aphid Rearing, Bacterial Infection and Hemocyte Phagocytosis Assay
    Authors:  Li Ma, Lu Liu and Zhiqiang Lu, date: 12/20/2020, view: 386, Q&A: 0
    [Abstract]

    Insects rely on the simple but effective innate immune system to combat infection. Cellular and humoral responses are interconnected and synergistic in insects’ innate immune system. Phagocytosis is one major cellular response. It is difficult to collect clean hemolymph from the small insect like pea aphid. Here, we provide a practicable

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    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: 657, Q&A: 0
    [Abstract]

    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

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    Preparation of Nippostrongylus brasiliensis Larvae for the Study of Host Skin Response
    Authors:  Tiffany Bouchery, Gillian Coakley and Nicola L. Harris, date: 12/20/2020, view: 359, Q&A: 0
    [Abstract]

    Hookworms are skin penetrating parasites, however in the laboratory the hookworm model Nippostrongylus brasiliensis, the parasite is traditionally administered subcutaneously bypassing the skin (epidermis and dermis). Here, we describe two complementary approaches for infecting mice with N. brasiliensis in order to study the skin immune responses.

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    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: 670, Q&A: 0
    [Abstract]

    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

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    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: 453, Q&A: 0
    [Abstract]

    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

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    Ribosome Purification from an α-proteobacterium and rRNA Analysis by Northern Blot
    Authors:  Liang Yin and Caroline S. Harwood, date: 12/05/2020, view: 405, Q&A: 0
    [Abstract] Ribosomes are an integral part of cellular life. They are complex molecular machines consisting of multiple ribosomal proteins and RNAs. To study different aspects of ribosome composition, many methods have been developed over the decades. Here, we describe how to purify ribosomes from the α-proteobacterium Rhodopseudomonas palustris ...
    Charging State Analysis of Transfer RNA from an α-proteobacterium
    Authors:  Liang Yin and Caroline S. Harwood, date: 12/05/2020, view: 389, Q&A: 0
    [Abstract] Transfer RNA (tRNA) is an essential link between the genetic code and proteins. During the process of translation, tRNA is charged with its cognate amino acid and delivers it to the ribosome, thus serving as a substrate of protein synthesis. To analyze the charging state of a particular tRNA, total RNA is purified and analyzed on an acid-urea gel. ...



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