Molecular Biology


    Protocols in Current Issue
    Experimental Models for Cold Exposure of Muscle in vitro and in vivo

    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

    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: 975, Q&A: 0

    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

    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: 1714, Q&A: 0

    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,

    Heterologous Expression and High Degree Purification of the Restriction Endonuclease SauUSI
    Authors:  Vinayak Sadasivam Tumuluri and Kayarat Saikrishnan, date: 01/05/2022, view: 2238, Q&A: 1

    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

    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: 1947, Q&A: 0

    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.

    Optimized Recombinant Production of Secreted Proteins Using Human Embryonic Kidney (HEK293) Cells Grown in Suspension

    Recombinant proteins are an essential milestone for a plethora of different applications ranging from pharmaceutical to clinical, and mammalian cell lines are among the currently preferred systems to obtain large amounts of proteins of interest due to their high level of post-translational modification and manageable large-scale production. In

    Affinity Purification of GO-Matryoshka Biosensors from E. coli for Quantitative Ratiometric Fluorescence Analyses
    [Abstract] Genetically encoded biosensors are powerful tools for quantitative visualization of ions and metabolites in vivo. Design and optimization of such biosensors typically require analyses of large numbers of variants. Sensor properties determined in vitro such as substrate specificity, affinity, response range, dynamic range, and ...
    High-level Production of Recombinant Membrane Proteins Using the Engineered Escherichia coli Strains SuptoxD and SuptoxR
    Authors:  Myrsini Michou, Dafni C. Delivoria and Georgios Skretas, date: 08/05/2020, view: 3503, Q&A: 0
    [Abstract] We have previously described the development of two specialized Escherichia coli strains for high-level recombinant membrane protein (MP) production. These engineered strains, termed SuptoxD and SuptoxR, are capable of suppressing the cytotoxicity caused by MP overexpression and of producing greatly enhanced MP yields. Here, we present a ...
    A Method for User-defined Mutagenesis by Integrating Oligo Pool Synthesis Technology with Nicking Mutagenesis
    Authors:  Paul J. Steiner, Zachary T Baumer and Timothy A. Whitehead, date: 08/05/2020, view: 4325, Q&A: 0
    [Abstract] Saturation mutagenesis is a fundamental enabling technology for protein engineering and epitope mapping. Nicking mutagenesis (NM) allows the user to rapidly construct libraries of all possible single mutations in a target protein sequence from plasmid DNA in a one-pot procedure. Briefly, one strand of the plasmid DNA is degraded using a nicking ...
    Expression and Purification of Functionally Active Serotonin 5-HT2A Receptor in Insect Cells Using Low-titer Viral Stock
    [Abstract] The serotonin 5-HT2A receptor (5-HT2AR) is a member of the GPCR family that is important for various neurological functions and whose dysregulation causes many mental health disorders. Structural investigations of 5-HT2AR require the production of functionally active receptors expressed from eukaryotic cell ...

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