Biochemistry

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    Protocols in Current Issue
    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: 359, 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: 243, 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: 357, 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: 365, 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: 828, 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: 681, 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: 381, 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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    Native Co-immunoprecipitation Assay to Identify Interacting Partners of Chromatin-associated Proteins in Mammalian Cells
    Authors:  Afzal Husain, Nasim A. Begum, Maki Kobayashi and Tasuku Honjo, date: 12/05/2020, view: 972, Q&A: 0
    [Abstract]

    Protein-protein interactions play key roles in nuclear processes including transcription, replication, DNA damage repair, and recombination. Co-immunoprecipitation (Co-IP) followed by western blot or mass spectrometry is an invaluable approach to identify protein-protein interactions. One of the challenges in the Co-IP of a protein localized to

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    Combining Gel Retardation and Footprinting to Determine Protein-DNA Interactions of Specific and/or Less Stable Complexes
    Authors:  Meng-Lun Hsieh, Alice Boulanger, Leslie G. Knipling and Deborah M. Hinton, date: 12/05/2020, view: 468, Q&A: 0
    [Abstract]

    DNA footprinting is a classic technique to investigate protein-DNA interactions. However, traditional footprinting protocols can be unsuccessful or difficult to interpret if the binding of the protein to the DNA is weak, the protein has a fast off-rate, or if several different protein-DNA complexes are formed. Our protocol differs from traditional

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    Cleavable Affinity Purification (Cl-AP): A One-step Procedure to Affinity Purify Protein Complexes
    Authors:  Ammarah Tariq, Lucy Green, Christian Soeller and James G. Wakefield, date: 11/20/2020, view: 1047, Q&A: 0
    [Abstract]

    Cleavable Affinity Purification (Cl-AP) uses a tripartite system of Protein-A-Streptavidin beads and nanobodies, coupled with a biotinylated, thiol-cleavable linker, providing one-step affinity purification from lysates of tissues expressing tagged proteins. This technique allows fluorescent versions of mitotic protein complexes to be isolated

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