Biochemistry

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    Protocols in Current Issue
    A Detailed Protocol for Preparing Millimeter-sized Supergiant Liposomes that Permit Efficient Eukaryotic Cell-free Translation in the Interior
    Authors:  Hajime Takahashi and Atsushi Ogawa, date: 06/20/2021, view: 581, Q&A: 0

    Liposomes have been used as a pseudo cell membrane for encapsulating biomolecules and creating an artificial cell in the interior where biochemical reactions can occur. Among the several methods used to prepare biomolecule-encapsulating liposomes,

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    Protein Import Assay into Mitochondria Isolated from Human Cells
    Authors:  Lena M. Murschall, Esra Peker, Thomas MacVicar, Thomas Langer and Jan Riemer, date: 06/20/2021, view: 945, Q&A: 0

    Mitochondria are essential organelles containing approximately 1,500 proteins. Only approximately 1% of these proteins are synthesized inside mitochondria, whereas the remaining 99% are synthesized as precursors on cytosolic ribosomes and imported

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    Enrichment of Vascular Fragments from Mouse Embryonic Brains for Endothelial Cell Analysis
    Authors:  Nicolas Santander and Thomas D. Arnold, date: 06/20/2021, view: 743, Q&A: 0

    Endothelial cells in the brain interact with other cell types, forming the blood-brain barrier. This barrier controls the movement of solutes into and out of the brain, regulating pathophysiological processes and drug delivery to the brain. Common

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    Fluorescence-based Heme Quantitation in Toxoplasma Gondii
    Authors:  Amy Bergmann and Zhicheng Dou, date: 06/20/2021, view: 656, Q&A: 0

    Toxoplasma gondii is a highly prevalent protozoan pathogen throughout the world. As a eukaryotic intracellular pathogen, Toxoplasma ingests nutrients from host cells to support its intracellular growth. The parasites also encode full or partial

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    Fluorescence-based Heme Quantitation in Toxoplasma Gondii
    Authors:  Amy Bergmann and Zhicheng Dou, date: 06/20/2021, view: 656, Q&A: 0
    [Abstract]

    Toxoplasma gondii is a highly prevalent protozoan pathogen throughout the world. As a eukaryotic intracellular pathogen, Toxoplasma ingests nutrients from host cells to support its intracellular growth. The parasites also encode full or partial metabolic pathways for the biosynthesis of certain nutrients, such as heme. Heme is an essential

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    Enrichment of Vascular Fragments from Mouse Embryonic Brains for Endothelial Cell Analysis
    Authors:  Nicolas Santander and Thomas D. Arnold, date: 06/20/2021, view: 743, Q&A: 0
    [Abstract]

    Endothelial cells in the brain interact with other cell types, forming the blood-brain barrier. This barrier controls the movement of solutes into and out of the brain, regulating pathophysiological processes and drug delivery to the brain. Common isolation methods used to study these cells during embryonic development involve enzymatic treatment

    ...
    Protein Import Assay into Mitochondria Isolated from Human Cells
    Authors:  Lena M. Murschall, Esra Peker, Thomas MacVicar, Thomas Langer and Jan Riemer, date: 06/20/2021, view: 945, Q&A: 0
    [Abstract]

    Mitochondria are essential organelles containing approximately 1,500 proteins. Only approximately 1% of these proteins are synthesized inside mitochondria, whereas the remaining 99% are synthesized as precursors on cytosolic ribosomes and imported into the organelle. Various tools and techniques to analyze the import process have been developed.

    ...
    A Detailed Protocol for Preparing Millimeter-sized Supergiant Liposomes that Permit Efficient Eukaryotic Cell-free Translation in the Interior
    Authors:  Hajime Takahashi and Atsushi Ogawa, date: 06/20/2021, view: 581, Q&A: 0
    [Abstract]

    Liposomes have been used as a pseudo cell membrane for encapsulating biomolecules and creating an artificial cell in the interior where biochemical reactions can occur. Among the several methods used to prepare biomolecule-encapsulating liposomes, the spontaneous emulsion transfer method is superior to others in that it allows us to readily

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    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: 1124, 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: 1595, 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: 1036, 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: 1310, 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: 1238, 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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