Biochemistry

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    Protocols in Current Issue
    Production of the Receptor-binding Domain of the Viral Spike Proteins from 2003 and 2019 SARS CoVs and the Four Common Human Coronaviruses for Serologic Assays and Inhibitor Screening
    [Abstract]

    The recombinant receptor-binding domain (RBD) of the viral spike protein from SARS-CoV-1 and 2 are reliable antigens for detecting viral-specific antibodies in humans. We and others have shown that the levels of RBD-binding antibodies and SARS-CoV-2 neutralizing antibodies in patients are correlated. Here, we report the expression and purification

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    A Fluorescence Dequenching-based Liposome Leakage Assay to Measure Membrane Permeabilization by Pore-forming Proteins
    Authors:  Javier Aguilera, Salvador Vazquez-Reyes and Jianjun Sun, date: 05/20/2021, view: 788, Q&A: 0
    [Abstract]

    Pore-forming toxins (PFTs) have been discovered in a wide range of organisms. Their functions are essential to the survival or virulence of many species. PFTs often interact with lipid membranes. Large unilamellar vesicles (LUV), also known as liposomes, have been commonly used as reliable membrane models for testing PFTs activity. Liposomes have

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    A New Method for Studying RNA-binding Proteins on Specific RNAs
    Authors:  Weiping Sun, Ziheng Zhang, Ji-Long Liu and Min Zhuang, date: 05/20/2021, view: 1147, Q&A: 0
    [Abstract]

    Proximity-based protein labeling has been developed to identify protein-nucleic acid interactions. We have reported a novel method termed CRUIS (CRISPR-based RNA-United Interacting System), which captures RNA-protein interactions in living cells by combining the RNA-binding capacity of CRISPR/Cas13 and the proximity-tagging activity of PUP-IT.

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    Intracellular IRF5 Dimerization Assay
    Authors:  Cherrie D. Sherman and Betsy J. Barnes, date: 05/20/2021, view: 821, Q&A: 0
    [Abstract]

    The intracellular interferon regulatory factor 5 (IRF5) dimerization assay is a technique designed to measure molecular interaction(s) with endogenous IRF5. Here, we present two methods that detect endogenous IRF5 homodimerization and interaction of endogenous IR5 with cell penetrating peptide (CPP) inhibitors. Briefly, to detect endogenous IRF5

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    Cell-free Synthesis of Correctly Folded Proteins with Multiple Disulphide Bonds: Production of Fungal Hydrophobins
    Authors:  Rezwan Siddiquee and Ann H Kwan, date: 05/20/2021, view: 1144, Q&A: 0
    [Abstract]

    Cell-free synthesis is a powerful technique that uses the transcriptional and translational machinery extracted from cells to create proteins without the constraints of living cells. Here, we report a cell-free protein production protocol using Escherichia coli lysate (Figure 1) to successfully express a class of proteins (known as hydrophobins)

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    Single-Molecule Studies of Membrane Receptors from Brain Region Specific Nanovesicles
    Authors:  Surya P. Aryal, Xu Fu, Abdullah A. Masud, Khaga R. Neupane and Christopher I. Richards, date: 05/20/2021, view: 728, Q&A: 0
    [Abstract]

    Single molecule imaging and spectroscopy are powerful techniques for the study of a wide range of biological processes including protein assembly and trafficking. However, in vivo single molecule imaging of biomolecules has been challenging because of difficulties associated with sample preparation and technical challenges associated with

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    Characterising Plant Deubiquitinases with in vitro Activity-based Labelling and Ubiquitin Chain Disassembly Assays
    Authors:  Michael J. Skelly and Steven H. Spoel, date: 05/05/2021, view: 412, Q&A: 0
    [Abstract]

    Post-translational modification of proteins by ubiquitin is an essential cellular signaling mechanism in all eukaryotes. Ubiquitin is removed from target proteins by a wide range of deubiquitinase (DUB) enzymes with different activities and substrate specificities. Understanding how DUBs function in vitro is a vital first step to uncovering their

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    Click Chemistry for Imaging in-situ Protein Palmitoylation during the Asexual Stages of Plasmodium falciparum
    Author:  Mansoor A. Siddiqui, date: 05/05/2021, view: 597, Q&A: 0
    [Abstract]

    Palmitoylation refers to the modification of the cysteine thiols in proteins by fatty acids, most commonly palmitic acid, through ‘thioester bond’ formation. In vivo, palmitoylation of proteins is catalyzed by palmitoyl acyltransferases (PATs or DHHC-PATs). Palmitoylation has recently emerged as a crucial post-translational

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    Proteomics Profiling of S-sulfurated Proteins in Acinetobacter baumannii
    Authors:  Brenna J. C. Walsh and David P. Giedroc, date: 05/05/2021, view: 406, Q&A: 0
    [Abstract]

    Hydrogen sulfide (H2S) is emerging as an important modulator in bacterial cytoprotection against the host immune response in infected animals, which may well be attributed to downstream highly oxidized sulfur species, termed reactive sulfur species (RSS), derived from H2S. One mechanism by which H2S/RSS may signal in the cell is through proteome

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    Optimized Recombinant Production of Secreted Proteins Using Human Embryonic Kidney (HEK293) Cells Grown in Suspension
    [Abstract]

    Recombinant proteins are an essential milestone for a plethora of different applications ranging from pharmaceutical to clinical, and mammalian cell lines are among the currently preferred systems to obtain large amounts of proteins of interest due to their high level of post-translational modification and manageable large-scale production. In

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