Biochemistry

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    Protocols in Current Issue
    Preparation of Cas9 Ribonucleoproteins for Genome Editing
    Authors:  Sheng-Wei Lin, Viet Quoc Nguyen and Steven Lin, date: 05/20/2022, view: 1340, 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

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    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

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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: 756, 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: 980, 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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    Measuring Oligonucleotide Hydrolysis in Cellular Lysates via Viscosity Measurements
    Authors:  Romel Menacho-Melgar and Michael D. Lynch, date: 01/20/2022, view: 1235, Q&A: 0
    [Abstract]

    Cell lysis, a process that releases host oligonucleotides, is required in many biotechnological applications. However, intact oligonucleotides in crude cellular lysates increase the viscosity of lysates, which complicates downstream processes and routine laboratory workflows. To address this, nucleases that hydrolyze the intact oligonucleotides

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    Isolation of Mitochondria from Ustilago maydis Protoplasts
    [Abstract]

    Ustilago maydis, a basidiomycete that infects Zea mays, is one of the top ten fungal models for studying DNA repair, signal transduction pathways, and dimorphic transitions, among other processes. From a metabolic point of view, U. maydis lacks fermentative capacity, pointing to mitochondria as a key player in central metabolism. Oxidative

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    Urea Denaturation, Zinc Binding, and DNA Binding Assays of Mutant p53 DNA-binding Domains and Full-length Proteins
    Authors:  Jeung-Hoi Ha, Xin Yu, Darren R. Carpizo and Stewart N. Loh, date: 10/20/2021, view: 1070, Q&A: 0
    [Abstract]

    In the cell, the thermodynamic stability of a protein – and hence its biological activity – can change dramatically as a result of perturbations in its amino acid sequence and the concentration of stabilizing ligands. This interplay is particularly evident in zinc-binding transcription factors such as the p53 tumor suppressor, whose

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    Measurement of the Translational Diffusion Coefficient and Hydrodynamic Radius of Proteins by Dynamic Light Scattering
    Authors:  Ernest T. Parker and Pete Lollar, date: 10/20/2021, view: 1511, Q&A: 0
    [Abstract]

    Diffusion is a fundamental process in biological systems that governs the molecular collisions driving biochemical reactions and membrane and transport. Measurement of the diffusion coefficient and application of the Stokes-Einstein equation produces the hydrodynamic radius, which is a commonly used gauge of particle size. Additionally,

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    Anaerobic Expression and Purification of Holo-CCIS, an Artificial Iron-sulfur Protein
    Authors:  Bhanu P. Jagilinki, Irina Paluy, Vikas Nanda and Dror Noy, date: 09/20/2021, view: 1608, Q&A: 0
    [Abstract]

    Iron-sulfur proteins are ubiquitous among all living organisms and are indispensable for almost all metabolic pathways ranging from photosynthesis, respiration, nitrogen, and carbon dioxide cycles. The iron-sulfur clusters primarily serve as electron acceptors and donors and transfer electrons to active sites of various enzymes, thus driving the

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    Methods for the Extraction of Heme Prosthetic Groups from Hemoproteins
    Authors:  Kat Ellis-Guardiola, Jess Soule and Robert T. Clubb, date: 09/20/2021, view: 1848, Q&A: 0
    [Abstract]

    Hemoproteins are widely researched because they contain redox-active heme prosthetic groups (iron + protoporphyrin IX) that enable them to perform a range of vital functions, acting as enzymes, participants in electron transfer reactions, or gas sensing, carrying, and storage proteins. While the heme prosthetic group is almost always essential for

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