Biochemistry

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    Protocols in Current Issue
    A mant-GDP Dissociation Assay to Compare the Guanine Nucleotide Binding Preference of Small GTPases
    Authors:  Yuping Tan and Qingxiang Sun, date: 01/20/2021, view: 98, Q&A: 0
    [Abstract]

    Small GTPases are cellular switches that are switched on when bound to GTP and switched off when bound to GDP. Different small GTPase proteins or those with mutations may bind to GTP or GDP with different relative affinities. However, small GTPases generally have very high affinities for guanine nucleotides, rendering it difficult to compare the

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    Polyamine Transport Assay Using Reconstituted Yeast Membranes
    Authors:  Sarah van Veen, Shaun Martin, Marleen Schuermans and Peter Vangheluwe, date: 01/20/2021, view: 128, Q&A: 0
    [Abstract]

    ATP13A2/PARK9 is a late endo-/lysosomal P5B transport ATPase that is associated with several neurodegenerative disorders. We recently characterized ATP13A2 as a lysosomal polyamine exporter, which sheds light on the molecular identity of the unknown mammalian polyamine transport system. Here, we describe step by step a protocol to measure

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    Reverse Phase-high-performance Liquid Chromatography (RP-HPLC) Analysis of Globin Chains from Human Erythroid Cells
    Authors:  Anne Chalumeau, Giacomo Frati, Elisa Magrin and Annarita Miccio, date: 01/20/2021, view: 89, Q&A: 0
    [Abstract]

    β-hemoglobinopathies are severe genetic disorders characterized either by the abnormal synthesis of the adult β-globin chains of the hemoglobin (Hb) tetramer (βS-globin chains) in sickle cell disease (SCD) or by the reduced β-globin production in β-thalassemia. The identification and quantification of globin chains are

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    Assessment of Diadenylate Cyclase and c-di-AMP-phosphodiesterase Activities Using Thin-layer and Ion Exchange Chromatography
    Authors:  Andreas Latoscha, David Jan Drexler, Gregor Witte and Natalia Tschowri, date: 01/05/2021, view: 379, Q&A: 0
    [Abstract]

    All living cells use cyclic nucleotides as second messengers for signal sensing and transduction. Cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) is primarily involved in the control of bacterial and euryarcheal osmoadaptation and is produced by diadenylate cyclases from two molecules of ATP. Specific phosphodiesterases hydrolyze

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    Non-separate Mouse Sclerochoroid/RPE/Retina Staining and Whole Mount for the Integral Observation of Subretinal Layer
    Authors:  Sung-Jin Lee and Soo-Young Kim, date: 01/05/2021, view: 254, Q&A: 0
    [Abstract]

    The subretinal layer between retinal pigment epithelium (RPE) and photoreceptors is a region involved in inflammation and angiogenesis during the procession of diseases such as age-related macular degeneration. The current protocols of whole mounts (retina and RPE) are unable to address the intact view of the subretinal layer because the

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    Non-radioactive Assay to Determine Product Profile of Short-chain Isoprenyl Diphosphate Synthases
    Authors:  Avanish Rai and Dinesh A. Nagegowda, date: 01/05/2021, view: 363, Q&A: 0
    [Abstract] Isoprenoids represent the largest class of metabolites with amazing diversities in structure and function. They are involved in protecting plants against pathogens or herbivores or involved in attracting pollinators. Isoprenoids are derived from geranyl diphosphate (GPP; C10), farnesyl diphosphate (FPP; C15), geranylgeranyl ...
    CENP-C Phosphorylation by CDK1 in vitro
    Authors:  Reito Watanabe, Masatoshi Hara, Mariko Ariyoshi and Tatsuo Fukagawa, date: 01/05/2021, view: 376, Q&A: 0
    [Abstract]

    Accurate chromosome segregation during mitosis requires the kinetochore, a large protein complex, which makes a linkage between chromosomes and spindle microtubes. An essential kinetochore component, CENP-C, is phosphorylated by Cyclin-B-Cyclin dependent kinase 1 (CDK1) that is a master kinase for mitotic progression, promoting proper kinetochore

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    A Quantitative Assay to Measure Stress Granule Association of Proteins and Peptides in Semi-permeabilized Human Cells
    Authors:  Saskia Hutten and Dorothee Dormann, date: 12/20/2020, view: 846, Q&A: 0
    [Abstract]

    Stress granules (SGs) are membrane-less organelles that form in the cytoplasm through phase separation, in response to diverse stressors. SGs contain translationally stalled mRNAs, proteins involved in translation, and various RNA-binding proteins (RBPs). Due to the high local concentration of aggregation-prone RBPs, SGs might act as condensation

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    Preparation of Bacterial Outer Membrane Vesicles for Characterisation of Periplasmic Proteins in Their Native Environment
    Authors:  Johannes Thoma and Björn M. Burmann, date: 12/20/2020, view: 698, Q&A: 0
    [Abstract]

    Bacterial outer membrane vesicles (OMVs) are naturally formed by budding from the outer membrane of Gram-negative bacteria. OMVs consist of a lipid bilayer identical in composition to the original outer membrane and contain periplasmic content within their lumen. Enriched with specific envelope proteins, OMVs make for an excellent native-like

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    Quantitative Irreversible Tethering (qIT) for Target-directed Covalent Fragment Screening
    Authors:  Gregory B. Craven, David J. Mann and Alan Armstrong, date: 12/20/2020, view: 390, Q&A: 0
    [Abstract]

    Small molecules that react to form covalent bonds with proteins are widely used as biological tools and therapeutic agents. Screening cysteine-reactive fragments against a protein target is an efficient way to identify chemical starting points for covalent probe development. Mass spectrometry is often used to identify the site and degree of

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