Biochemistry

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    Expression and Purification of Arabidopsis Transmembrane Protein BCM1 in Saccharomyces cerevisiae
    Authors:  Peng Wang and Bernhard Grimm, date: 09/20/2020, view: 84, Q&A: 0
    [Abstract] Heterologous expression and purification of transmembrane proteins have remained a challenge for decades hampering detailed biochemical and structural characterization of key enzymes and their interacting regulators in multiple metabolic pathways. An in-depth study on the newly identified Arabidopsis thaliana integral membrane protein ...
    Novel Protein-oligonucleotide Conjugation Method Involving a High-affinity Capture HaloTag
    Author:  Junshi Yazaki, date: 09/20/2020, view: 99, Q&A: 0
    [Abstract] Highly sensitive quantitative protein profiling can play a key role in the early diagnosis of diseases, such as autoimmune diseases and cancer. We developed a modified protein-oligonucleotide conjugation method termed HaloTag-mediated barcoding, for quantifying protein molecules at a higher sensitivity than conventional protein quantification ...
    In vitro AMPylation/Adenylylation of Alpha-synuclein by HYPE/FICD
    Authors:  Ali Camara, Anwesha Sanyal, Sayan Dutta, Jean-Christophe Rochet and Seema Mattoo, date: 09/20/2020, view: 85, Q&A: 0
    [Abstract] One of the major histopathological hallmarks of Parkinson’s disease are Lewy bodies (LBs) –cytoplasmic inclusions, enriched with fibrillar forms of the presynaptic protein alpha-synuclein (α-syn). Progressive deposition of α-syn into LBs is enabled by its propensity to fibrillize into insoluble aggregates. We recently described a marked reduction ...
    A Workflow for Ultra-rapid Analysis of Histone Post-translational Modifications with Direct-injection Mass Spectrometry
    Authors:  Natarajan V. Bhanu, Simone Sidoli and Benjamin A Garcia, date: 09/20/2020, view: 102, Q&A: 0
    [Abstract] Chromatin modifications, like histone post translational modifications (PTMs), are critical for tuning gene expression and many other aspects of cell phenotype. Liquid chromatography coupled to mass spectrometry (LC-MS) has become the most suitable method to analyze histones and histone PTMs in a large-scale manner. Selected histone PTMs have ...
    Optogenetic Tuning of Protein-protein Binding in Bilayers Using LOVTRAP
    Authors:  Doug Tischer and Orion D. Weiner, date: 09/05/2020, view: 696, Q&A: 0
    [Abstract] Modern microscopy methods are powerful tools for studying live cell signaling and biochemical reactions, enabling us to observe when and where these reactions take place from the level of a cell down to single molecules. With microscopy, each cell or molecule can be observed both before and after a given perturbation, facilitating better inference ...
    Karyopherin-β2 Inhibits and Reverses Aggregation and Liquid-liquid Phase Separation of the ALS/FTD-Associated Protein FUS
    Authors:  Emma Robinson, James Shorter and Lin Guo, date: 08/20/2020, view: 776, Q&A: 0
    [Abstract] The study of RNA-binding proteins (RBP) offers insight into the mechanisms of pathologic protein aggregation in neurodegenerative diseases. We developed a protocol for purifying an RBP FUS and a nuclear import receptor (NIR) Kapβ2 and testing the ability of Kapβ2 to mitigate FUS aggregation and liquid-liquid phase separation.
    Microtubule Seeded-assembly in the Presence of Poorly Nucleating Nucleotide Analogues
    Authors:  Siou Ku, Claire Heichette, Laurence Duchesne and Denis Chrétien, date: 08/20/2020, view: 473, Q&A: 0
    [Abstract] Microtubule dynamic instability is driven by the hydrolysis of the GTP bound to the β-subunit of the α-β tubulin heterodimer. Nucleotide analogues are commonly used to mimic the different steps of the tubulin GTPase cycle, but most of them are poor microtubule nucleators. Usually, microtubule assembly is seeded by guanylyl-(α, ...
    The ATPase Activity of Escherichia coli Expressed AAA+-ATPase Protein
    Authors:  Amita Kaundal, Vemanna S. Ramu and Kirankumar S. Mysore, date: 08/05/2020, view: 584, Q&A: 0
    [Abstract] ATPases are the enzymes that breakdown ATP to ADP and release inorganic phosphate (Pi). Here we provide a detailed protocol to determine the ATPase activity of a recombinant AAA+-ATPase protein (GENERAL CONTROL NON-REPRESSIBLE-4 [GCN4]) by spectrophotometric absorption at 360 nm to measure the accumulated inorganic phosphate. In ...
    A Method for User-defined Mutagenesis by Integrating Oligo Pool Synthesis Technology with Nicking Mutagenesis
    Authors:  Paul J. Steiner, Zachary T Baumer and Timothy A. Whitehead, date: 08/05/2020, view: 823, Q&A: 0
    [Abstract] Saturation mutagenesis is a fundamental enabling technology for protein engineering and epitope mapping. Nicking mutagenesis (NM) allows the user to rapidly construct libraries of all possible single mutations in a target protein sequence from plasmid DNA in a one-pot procedure. Briefly, one strand of the plasmid DNA is degraded using a nicking ...
    Expression and Purification of Functionally Active Serotonin 5-HT2A Receptor in Insect Cells Using Low-titer Viral Stock
    [Abstract] The serotonin 5-HT2A receptor (5-HT2AR) is a member of the GPCR family that is important for various neurological functions and whose dysregulation causes many mental health disorders. Structural investigations of 5-HT2AR require the production of functionally active receptors expressed from eukaryotic cell ...



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