Biochemistry

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    Protocols in Current Issue
    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: 1988, 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

    ...
    FRET-based Microscopy Assay to Measure Activity of Membrane Amino Acid Transporters with Single-transporter Resolution
    [Abstract]

    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

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    Conjugation of Fab’ Fragments with Fluorescent Dyes for Single-molecule Tracking on Live Cells
    Authors:  I-Ting Teng, Xiangning Bu and Inhee Chung, date: 09/20/2019, view: 3753, Q&A: 0
    [Abstract] Our understanding of the regulation and functions of cell-surface proteins has progressed rapidly with the advent of advanced optical imaging techniques. In particular, single-molecule tracking (SMT) using bright fluorophores conjugated to antibodies and wide-field microscopy methods such as total internal reflection fluorescence microscopy have ...
    Fluorophore Labeling, Nanodisc Reconstitution and Single-molecule Observation of a G Protein-coupled Receptor
    Authors:  Rajan Lamichhane, Jeffrey J. Liu, Raymond F. Pauszek III and David P. Millar, date: 06/20/2017, view: 7186, Q&A: 0
    [Abstract] Activation of G protein-coupled receptors (GPCRs) by agonist ligands is mediated by a transition from an inactive to active receptor conformation. We describe a novel single-molecule assay that monitors activation-linked conformational transitions in individual GPCR molecules in real-time. The receptor is site-specifically labeled with a Cy3 ...
    Cloud-point PEG Glass Surfaces for Imaging of Immobilized Single Molecules by Total-internal-reflection Microscopy
    Authors:  Zhengjian Zhang, Sang Ryul Park, Alexandros Pertsinidis and Andrey Revyakin, date: 04/05/2016, view: 9244, Q&A: 0
    [Abstract] This effective, robust protocol generates glass coverslips coated with biotin-functionalized polyethylene glycol (PEG), making the glass surface resistant to non-specific absorption of biomolecules, and permitting immobilization of biomolecules for subsequent single-molecule tracking of biochemical reactions. The protocol can be completed in one ...



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