Protocols in Current Issue
    A High-throughput Pipeline to Determine DNA and Nucleosome Conformations by AFM Imaging
    Authors:  Sebastian F. Konrad, Willem Vanderlinden and Jan Lipfert, date: 10/05/2021, view: 460, Q&A: 0

    Atomic force microscopy (AFM) is a powerful tool to image macromolecular complexes with nanometer resolution and exquisite single-molecule sensitivity. While AFM imaging is well-established to investigate DNA and nucleoprotein complexes, AFM studies are often limited by small datasets and manual image analysis that is slow and prone to user bias.

    Using Atomic Force Microscopy to Study the Real Time Dynamics of DNA Unwinding by Mitochondrial Twinkle Helicase
    Authors:  Parminder Kaur, Hai Pan, Matthew J. Longley, William C. Copeland and Hong Wang, date: 09/05/2021, view: 943, Q&A: 0

    Understanding the structure and dynamics of DNA-protein interactions during DNA replication is crucial for elucidating the origins of disorders arising from its dysfunction. In this study, we employed Atomic Force Microscopy as a single-molecule imaging tool to examine the mitochondrial DNA helicase Twinkle and its interactions with DNA. We used

    Unraveling the Physicochemical Determinants of Protein Liquid-liquid Phase Separation by Nanoscale Infrared Vibrational Spectroscopy
    Authors:  Francesco S. Ruggeri, Alyssa M. Miller, Michele Vendruscolo and Tuomas P. J. Knowles, date: 08/20/2021, view: 1388, Q&A: 0

    The phenomenon of reversible liquid-liquid phase separation of proteins underlies the formation of membraneless organelles, which are crucial for cellular processes such as signalling and transport. In addition, it is also of great interest to uncover the mechanisms of further irreversible maturation of the functional dense liquid phase into

    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: 2570, 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,

    Purification of Rice Stripe Virus
    Authors:  Gang Lu, Min Yao, Yijun Zhou and Xiaorong Tao, date: 03/20/2020, view: 1974, 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 ...
    Studying Protein Aggregation in the Context of Liquid-liquid Phase Separation Using Fluorescence and Atomic Force Microscopy, Fluorescence and Turbidity Assays, and FRAP
    Authors:  W. Michael Babinchak and Witold K. Surewicz, date: 01/20/2020, view: 4391, Q&A: 0
    [Abstract] Liquid-liquid phase separation (LLPS) underlies the physiological assembly of many membrane-less organelles throughout the cell. However, dysregulation of LLPS may mediate the formation of pathological aggregates associated with neurodegenerative diseases. Here, we present complementary experimental approaches to study protein aggregation within ...

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