Protocols in Current Issue
    FRET-based Microscopy Assay to Measure Activity of Membrane Amino Acid Transporters with Single-transporter Resolution

    Secondary active transporters reside in cell membranes transporting polar solutes like amino acids against steep concentration gradients, using electrochemical gradients of ions as energy sources. Commonly, ensemble-based measurements of radiolabeled substrate uptakes or transport currents inform on kinetic parameters of transporters. Here we

    A Workflow for High-pressure Freezing and Freeze Substitution of the Caenorhabditis elegans Embryo for Ultrastructural Analysis by Conventional and Volume Electron Microscopy
    Authors:  Mohammad M. Rahman, Irene Y. Chang, Orna Cohen-Fix and Kedar Narayan, date: 04/05/2021, view: 182, Q&A: 0

    The free-living nematode Caenorhabditis elegans is a popular model system for studying developmental biology. Here we describe a detailed protocol to high-pressure freeze the C. elegans embryo (either ex vivo after dissection, or within the intact worm) followed by quick freeze substitution. Processed samples are suitable for ultrastructural

    Automated Analysis of Cerebrospinal Fluid Flow and Motile Cilia Properties in The Central Canal of Zebrafish Embryos

    Circulation of cerebrospinal fluid (CSF) plays an important role during development. In zebrafish embryo, the flow of CSF has been found to be bidirectional in the central canal of the spinal cord. In order to compare conditions and genetic mutants across each other, we recently automated the quantification of the velocity profile of exogenous

    Characterize the Interaction of the DNA Helicase PriA with the Stalled DNA Replication Fork Using Atomic Force Microscopy
    Authors:  Yaqing Wang, Zhiqiang Sun, Piero R. Bianco and Yuri L. Lyubchenko, date: 03/05/2021, view: 967, Q&A: 0

    In bacteria, the restart of stalled DNA replication forks requires the DNA helicase PriA. PriA can recognize and remodel abandoned DNA replication forks, unwind DNA in the 3'-to-5' direction, and facilitate the loading of the helicase DnaB onto the DNA to restart replication. ssDNA-binding protein (SSB) is typically present at the abandoned forks,

    Mechanical Characterization of Glandular Acini Using a Micro-indentation Instrument
    Authors:  Christopher S. O’Bryan, Qiao Zhang, Tanmay P. Lele and Thomas E. Angelini, date: 12/05/2020, view: 835, Q&A: 0

    The linker of nucleoskeleton and cytoskeleton (LINC) complex is responsible for tethering the nucleus to the cytoskeleton, providing a pathway for the cell’s nucleus to sense mechanical signals from the environment. Recently, we explored the role of the LINC complex in the development of glandular epithelial acini, such as those found in

    Assembly and Imaging Set up of PIE-Scope
    [Abstract] Cryo-Electron Tomography (cryo-ET) is a method that enables resolving the structure of macromolecular complexes directly in the cellular environment. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-Focused Ion Beam (FIB) milling and correlative light microscopy ...
    Optogenetic Tuning of Protein-protein Binding in Bilayers Using LOVTRAP
    Authors:  Doug Tischer and Orion D. Weiner, date: 09/05/2020, view: 2192, 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 ...
    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 ...
    Purification of Rice Stripe Virus
    Authors:  Gang Lu, Min Yao, Yijun Zhou and Xiaorong Tao, date: 03/20/2020, view: 1407, Q&A: 0
    [Abstract] Although many spherical and rod-shaped plant virus purification protocols are now available, only a few protocols on filamentous plant virus purification have been published. Here, we report a protocol for large-scale purification of Rice stripe virus (RSV) from RSV-infected rice tissues. RSV virions with high infectivity were first precipitated ...
    Protocols for Processing and Interpreting cryoEM Data Using Bsoft: A Case Study of the Retinal Adhesion Protein, Retinoschisin
    Author:  J. Bernard Heymann, date: 01/20/2020, view: 2154, Q&A: 0
    [Abstract] The goal of cryoEM is to determine the structures of biomolecules from electron micrographs. In many cases the processing is straightforward and can be handled with routine protocols. In other cases, the properties and behavior of the specimen require adaptions to properly interpret the data. Here I describe the protocols for examining the higher ...

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