Molecular Biology

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    Protocols in Current Issue
    DSP-crosslinking and Immunoprecipitation to Isolate Weak Protein Complex
    Authors:  Kotaro Akaki, Takashi Mino and Osamu Takeuchi, date: 08/05/2022, view: 145, Q&A: 0
    [Abstract]

    Detecting protein-protein interactions (PPIs) is one of the most used approaches to reveal the molecular regulation of protein of interests (POIs). Immunoprecipitation of POIs followed by mass spectrometry or western blot analysis enables us to detect co-precipitated POI-binding proteins. However, some binding proteins are lost during cell lysis

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    Experimental Models for Cold Exposure of Muscle in vitro and in vivo
    [Abstract]

    Work in cold environments may have a significant impact on occupational health. In these and similar situations, cold exposure localized to the extremities may reduce the temperature of underlying tissues. To investigate the molecular effects of cold exposure in muscle, and since adequate methods were missing, we established two experimental cold

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    Application of Cadaverine to Inhibit Biotin Biosynthesis in Plants
    Authors:  Nicole M. Gibbs, Shih-Heng Su and Patrick H. Masson, date: 04/20/2022, view: 671, Q&A: 0
    [Abstract]

    Biotin is an essential vitamin in plants. However, characterization of biotin deficiency has been limited by embryo lethality in mutants, which can only be rescued by supplementation of biotin. Here, we describe a protocol to characterize biotin deficiency in Arabidopsis thaliana through application of the polyamine cadaverine. Cadaverine

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    Colocalization Analysis for Cryosectioned and Immunostained Tissue Samples with or without Label Retention Expansion Microscopy (LR-ExM) by JACoP
    Authors:  Xiang Zhao, Jason Garcia, Loic A. Royer and Su Guo, date: 03/05/2022, view: 2188, Q&A: 0
    [Abstract]

    Asymmetric cell division (ACD) is fundamental for balancing cell proliferation and differentiation in metazoans. During active neurogenesis in the developing zebrafish forebrain, radial glia progenitors (RGPs) mainly undergo ACD to produce one daughter with high activity of Delta/Notch signaling (proliferative cell fate) and another daughter with

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    Simple Scalable Protein Expression and Extraction Using Two-stage Autoinducible Cell Autolysis and DNA/RNA Autohydrolysis in Escherichia coli
    Authors:  Romel Menacho-Melgar and Michael D. Lynch, date: 01/20/2022, view: 1309, Q&A: 0
    [Abstract]

    Recombinant protein expression is extensively used in biological research. Despite this, current protein expression and extraction methods are not readily scalable or amenable for high-throughput applications. Optimization of protein expression conditions using traditional methods, reliant on growth-associated induction, is non-trivial. Similarly,

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    A High-throughput Automated ELISA Assay for Detection of IgG Antibodies to the SARS-CoV-2 Spike Protein
    [Abstract]

    The SARS-CoV-2 pandemic and vaccination campaign has illustrated the need for high throughput serological assays to quantitatively measure antibody levels. Here, we present a protocol for a high-throughput colorimetric ELISA assay to detect IgG antibodies against the SARS-CoV-2 spike protein. The assay robustly distinguishes positive from negative

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    Heterologous Expression and High Degree Purification of the Restriction Endonuclease SauUSI
    Authors:  Vinayak Sadasivam Tumuluri and Kayarat Saikrishnan, date: 01/05/2022, view: 1846, Q&A: 1
    [Abstract]

    Mechanisms that target and destroy foreign nucleic acids are major barriers to horizontal gene transfer (HGT) in prokaryotes. Amongst them, restriction-modification (R-M) systems are found in ≥75% of the sequenced genomes in Bacteria and Archaea. Due to their high target sequence specificity and potent nucleolytic activity, R-M systems are used

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    Ex-vivo Microtubule Stability Assay Using Drosophila Wing Disc
    Authors:  Jung-Wan Mok and Kwang-Wook Choi, date: 12/05/2021, view: 1229, Q&A: 0
    [Abstract]

    Regulation of microtubule stability is crucial for diverse biological processes, including cell division, morphogenesis, and signaling. Various in vitro assays for microtubule stability have been developed to identify and characterize proteins involved in controlling microtubule stability. Here, we introduce a simple ex-vivo assay for identifying

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    Knockoff: Druggable Cleavage of Membrane Proteins
    Authors:  John H. Rinald, Jason D. Vevea and Edwin R. Chapman, date: 11/20/2021, view: 1354, Q&A: 0
    [Abstract]

    Comparative cell biology relies on methods that disrupt protein function. Traditional approaches target the gene that encodes the protein of interest via conventional knockout (KO) methodology, conditional Cre-lox system, or recently, flexible protocols based on CRISPR/Cas9. However, these technologies lack precise temporal control (hours),

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    Co-immunofluorescence of MRPL12 and Nrf2 in HK2 Cells
    Authors:  Yao Lu, Xia Gu, Qiang Wan, Hong Feng and Yi Liu, date: 10/20/2021, view: 1504, Q&A: 0
    [Abstract]

    Immunofluorescence is a technique to visualize the localization of specific molecule targets within cells using the specificity of antibodies. Here, we describe a protocol to detect two different protein components in a cell simultaneously. Antibody concentrations to be used vary from cell to cell and should be optimized for different cell types.

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