Molecular Biology

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    Protocols in Current Issue
    A Novel Imaging Technique for The On-site Assessment of Renal Biopsy Specimens
    Authors:  Tomoaki Takata, Hajime Isomoto, Takuji Iyama and Kentaro Yamada, date: 09/20/2022, view: 266, Q&A: 0
    [Abstract]

    When performing renal biopsy, it is necessary to identify the cortex, where glomeruli are exclusively distributed, to ensure the quality of the specimen for histological diagnosis. However, conventional methods using a stereomicroscope or magnifying lens often fail to clarify the quality of the specimen. We have established a fluorescent-based

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    In-Cell Western Protocol for Semi-High-Throughput Screening of Single Clones
    Authors:  Arpita S. Pal, Alejandra M. Agredo and Andrea L. Kasinski, date: 08/20/2022, view: 1145, Q&A: 0
    [Abstract]

    The in-cell western (ICW) is an immunocytochemical technique that has been used to screen for effects of siRNAs, drugs, and small molecule inhibitors. The reduced time and number of cells required to follow this protocol illustrates its semi-high-throughput nature. Performing a successful ICW protocol requires fixing and permeabilizing adherent

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    Colocalization Assay with Fluorescent-tagged ATG8 Using a Nicotiana benthamiana-based Transient System
    Authors:  Jinyan Mai, Dandan Shang, Faqiang Li and Na Luo, date: 08/20/2022, view: 711, Q&A: 0
    [Abstract]

    Autophagy is an evolutionarily conserved intracellular degradation process. During autophagy, a set of autophagy-related (ATG) proteins orchestrate the formation of double-bound membrane vesicles called autophagosomes to engulf cytoplasmic material and deliver it to the vacuole for breakdown. Among ATG proteins, the ATG8 is the only one decorating

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    DSP-crosslinking and Immunoprecipitation to Isolate Weak Protein Complex
    Authors:  Kotaro Akaki, Takashi Mino and Osamu Takeuchi, date: 08/05/2022, view: 623, 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: 784, 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: 2394, 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: 1501, 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: 2011, 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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