Biochemistry

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    Protocols in Current Issue
    In vitro Assays to Evaluate Specificity and Affinity in Protein-phospholipid Interactions

    Protein–lipid interactions play important roles in many biological processes, including metabolism, signaling, and transport; however, computational and structural analyses often fail to predict such interactions, and determining which lipids

    ...
    Preparation of Cas9 Ribonucleoproteins for Genome Editing
    Authors:  Sheng-Wei Lin, Viet Quoc Nguyen and Steven Lin, date: 05/20/2022, view: 36, Q&A: 0

    Genome editing by the delivery of pre-assembled Cas9 ribonucleoproteins (Cas9 RNP) is an increasingly popular approach for cell types that are difficult to manipulate genetically by the conventional plasmid and viral methods. Cas9 RNP editing is

    ...
    Identification and Monitoring of Nucleotide Repeat Expansions Using Southern Blotting in Drosophila Models of C9orf72 Motor Neuron Disease and Frontotemporal Dementia
    Authors:  Joanne L. Sharpe, Nikki S. Harper and Ryan J. H. West, date: 05/20/2022, view: 24, Q&A: 0

    Repeat expansion diseases, including fragile X syndrome, Huntington’s disease, and C9orf72-related motor neuron disease and frontotemporal dementia, are a group of disorders associated with polymorphic expansions of tandem repeat nucleotide

    ...
    Purification of the Bacterial Amyloid “Curli” from Salmonella enterica Serovar Typhimurium and Detection of Curli from Infected Host Tissues

    Microbiologists are learning to appreciate the importance of “functional amyloids” that are produced by numerous bacterial species and have impacts beyond the microbial world. These structures are used by bacteria to link together,

    ...
    In vitro Assays to Evaluate Specificity and Affinity in Protein-phospholipid Interactions
    [Abstract]

    Protein–lipid interactions play important roles in many biological processes, including metabolism, signaling, and transport; however, computational and structural analyses often fail to predict such interactions, and determining which lipids participate in these interactions remains challenging. In vitro assays to assess the physical

    ...
    Preparation of Cas9 Ribonucleoproteins for Genome Editing
    Authors:  Sheng-Wei Lin, Viet Quoc Nguyen and Steven Lin, date: 05/20/2022, view: 36, Q&A: 0
    [Abstract]

    Genome editing by the delivery of pre-assembled Cas9 ribonucleoproteins (Cas9 RNP) is an increasingly popular approach for cell types that are difficult to manipulate genetically by the conventional plasmid and viral methods. Cas9 RNP editing is robust, precise, capable of multiplexing, and free of genetic materials. Its transient presence in

    ...
    Identification and Monitoring of Nucleotide Repeat Expansions Using Southern Blotting in Drosophila Models of C9orf72 Motor Neuron Disease and Frontotemporal Dementia
    Authors:  Joanne L. Sharpe, Nikki S. Harper and Ryan J. H. West, date: 05/20/2022, view: 24, Q&A: 0
    [Abstract]

    Repeat expansion diseases, including fragile X syndrome, Huntington’s disease, and C9orf72-related motor neuron disease and frontotemporal dementia, are a group of disorders associated with polymorphic expansions of tandem repeat nucleotide sequences. These expansions are highly repetitive and often hundreds to thousands of repeats in

    ...
    Purification of the Bacterial Amyloid “Curli” from Salmonella enterica Serovar Typhimurium and Detection of Curli from Infected Host Tissues
    [Abstract]

    Microbiologists are learning to appreciate the importance of “functional amyloids” that are produced by numerous bacterial species and have impacts beyond the microbial world. These structures are used by bacteria to link together, presumably to increase survival, protect against harsh conditions, and perhaps to influence cell-cell

    ...
    Production and Crystallization of Nanobodies in Complex with the Receptor Binding Domain of the SARS-CoV-2 Spike Protein
    [Abstract]

    The receptor binding domain (RBD) of the spike protein of SARS-CoV-2 binds angiotensin converting enzyme-2 (ACE-2) on the surface of epithelial cells, leading to fusion, and entry of the virus into the cell. This interaction can be blocked by the binding of llama-derived nanobodies (VHHs) to the RBD, leading to virus neutralisation. Structural

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    Translating Ribosome Affinity Purification (TRAP) of Cell Type-specific mRNA from Mouse Brain Lysates
    Authors:  Catherine L. Salussolia, Kellen D. Winden and Mustafa Sahin, date: 05/05/2022, view: 438, Q&A: 0
    [Abstract]

    Mammalian tissues are highly heterogenous and complex, posing a challenge in understanding the molecular mechanisms regulating protein expression within various tissues. Recent studies have shown that translation at the level of the ribosome is highly regulated, and can vary independently of gene expression observed at a transcriptome level, as

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    Immunoisolation of Endosomal Recycling Vesicles from Saccharomyces cerevisiae
    Authors:  Sho W. Suzuki and Scott D. Emr, date: 05/05/2022, view: 440, Q&A: 0
    [Abstract]

    Endosomal recycling is essential for the appropriate function of the endosome. During this process, endosomal coat complexes (i.e., retromer, and Mvp1) are recruited to the endosome, and deform its membrane to form recycling vesicles. To further analyze this, we developed a protocol for the immunoisolation of recycling vesicles from budding yeast.

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    Expression, Purification, and in vitro Enzyme Activity Assay of a Recombinant Aldehyde Dehydrogenase from Thermus thermophilus, using an Escherichia coli host
    Authors:  Kim Shortall, Edmond Magner and Tewfik Soulimane, date: 05/05/2022, view: 552, Q&A: 0
    [Abstract]

    Based on previous in-depth characterisation, aldehyde dehydrogenases (ALDH) are a diverse superfamily of enzymes, in terms of both structure and function, present in all kingdoms of life. They catalyse the oxidation of an aldehyde to carboxylic acid using the cofactor nicotinamide adenine dinucleotide (phosphate) (NAD(P)+), and are often not

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    Apoplastic Expression of CARD1-ecto Domain in Nicotiana benthamiana and Purification from the Apoplastic Fluids
    Authors:  Nobuaki Ishihama, Anuphon Laohavisit, Kaori Takizawa and Ken Shirasu, date: 04/20/2022, view: 767, Q&A: 0
    [Abstract]

    The protein expression and purification process is an essential initial step for biochemical analysis of a protein of interest. Traditionally, heterologous protein expression systems (such as E. coli, yeast, insect cells, and cell-free) are employed for plant protein expression, although a plant expression system is often desirable for plant

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    Visualization and Purification of Caenorhabditis elegans Germ Granule Proteins Using Proximity Labeling
    Authors:  Hannah L. Hertz, Ian F. Price and Wen Tang, date: 04/20/2022, view: 1038, Q&A: 0
    [Abstract]

    Membraneless organelles, such as germ granules and stress granules, are liquid-like condensates formed by phase transition. Recently, we and others have adopted proximity-based labeling methods to determine the composition of these membraneless compartments. Here, we describe the use of TurboID—an engineered promiscuous biotin

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