Biochemistry

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    Protocols in Current Issue
    A SYBR Gold-based Label-free in vitro Dicing Assay
    [Abstract]

    In Arabidopsis, DICER-LIKE PROTEIN 3 (DCL3) cuts the substrate pre-siRNA into a product siRNA duplex, encompassing one 23-nt strand and one 24-nt strand. To monitor the separation of the siRNA duplex with only 1-nt difference, we developed this protocol to evaluate the in vitro dicing activity of DCL3. The method can be applied for measuring

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    In vitro Auto- and Substrate-Ubiquitination Assays
    Authors:  Hye Lin Park, Han Yong Lee and Gyeong Mee Yoon, date: 04/05/2022, view: 582, Q&A: 0
    [Abstract]

    The precise regulation of the homeostasis of the cellular proteome is critical for the appropriate growth and development of plants. It also allows the plants to respond to various environmental stresses, by modulating their biochemical and physiological aspects in a timely manner. Ubiquitination of cellular proteins is one of the major

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    Tracking the Reversed Oxidative Tricarboxylic Acid Cycle in Bacteria
    [Abstract]

    Different pathways for autotrophic CO2 fixation can be recognized by the presence of genes for their specific key enzymes. On this basis, (meta)genomic, (meta)transcriptomic, or (meta)proteomic analysis enables the identification of the role of an organism or a distinct pathway in primary production. However, the recently discovered variant of the

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    Activity-based Protein Profiling of Serine Hydrolase Superfamily Enzymes
    Authors:  Achintya Kumar Dolui, Mahadev Latha and Panneerselvam Vijayaraj, date: 03/20/2022, view: 624, Q&A: 0
    [Abstract]

    Activity-based protein profiling (ABPP) is a chemoproteomics platform to assess the functional state of enzymes in complex biological systems. Over the two decades, ABPP has emerged from a gel-based to gel-free platform, for in-depth proteome analysis with enhanced resolution, sensitivity for target detection, and discovery of small molecule

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    A Simple Technique for Direct Immobilization of Target Enzymes from Cell Lysates Based on the SpyTag/SpyCatcher Spontaneous Reaction
    Authors:  Li-xi Cai, Yuan-qing Lin, Yun-meng Chu, Xiao-pin Chen, Li-xing Liu, Ming Zhang and Guang-ya Zhang, date: 01/05/2022, view: 1594, Q&A: 0
    [Abstract]

    Many of the current methods for enzyme purification and immobilization suffer from several drawbacks, such as requiring tedious multistep procedures or long preparation, and being environmentally unfriendly, due to the chemicals and conditions involved. Thus, a simple technique for direct purification and immobilization of target enzymes from cell

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    An in vitro Coupled Assay for PEPC with Control of Bicarbonate Concentration
    Authors:  Nicholas R. Moody, Chatawal Phansopal and James D. Reid, date: 12/20/2021, view: 888, Q&A: 0
    [Abstract]

    Phosphoenolpyruvate carboxylase (PEPC) catalyzes a critical step in carbon metabolism in plants and bacteria, the irreversible reaction between bicarbonate and phosphoenolpyruvate to produce the C4 compound oxaloacetate. This enzyme is particularly important in the context of C4 photosynthesis, where it is the initial carbon-fixing enzyme. Many

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    An Assay to Determine NAD(P)H: Quinone Oxidoreductase Activity in Cell Extracts from Candida glabrata
    Authors:  Anamika Battu, Rajaram Purushotham and Rupinder Kaur, date: 11/05/2021, view: 1095, Q&A: 0
    [Abstract]

    Flavodoxin-like proteins (Fld-LPs) are an important constituent of the oxidative stress defense system in several organisms and highly conserved from bacteria to humans. These proteins possess NAD(P)H:quinone oxidoreductase activity and convert quinones to hydroquinones through two-electron reduction, using NAD(P)H and quinone as electron donor

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    Measuring Real-time DNA/RNA Nuclease Activity through Fluorescence
    Authors:  Paulina Wyrzykowska, Sally Rogers and Richard Chahwan, date: 11/05/2021, view: 1820, Q&A: 0
    [Abstract]

    DNA and RNA nucleases are wide-ranging enzymes, taking part in broad cellular processes from DNA repair to immune response control. Growing interest in the mechanisms and activities of newly discovered nucleases inspired us to share the detailed protocol of our nuclease assay (Sheppard et al., 2019). This easy and inexpensive method can provide

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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: 1042, 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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    High Throughput Analyses of Ascorbate-turnover Enzyme Activities in Rice (Oryza sativa L.) Seedlings
    Authors:  Lin-Bo Wu, Yanru Feng, Frederike Zeibig, Muhammad Shahedul Alam and Michael Frei, date: 10/20/2021, view: 1308, Q&A: 0
    [Abstract]

    Ascorbate (Vitamin C) fulfills various functions in plant photosynthesis and abiotic stress tolerance. The four key enzymes involved in the ascorbate-turnover pathway are ascorbate peroxidase, ascorbate oxidase, monodehydroascorbate reductase, and dehydroascorbate reductase. Several reports have shown the pivotal roles of these enzymes in plant

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