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

    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: 3267, 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 ...
    Preparation of Cell-free Synthesized Proteins Selectively Double Labeled for Single-molecule FRET Studies
    Authors:  Mayuri Sadoine, Michele Cerminara, Jörg Fitter and Alexandros Katranidis, date: 06/20/2018, view: 4022, Q&A: 0
    [Abstract] Single-molecule FRET (smFRET) is a powerful tool to investigate molecular structures and conformational changes of biological molecules. The technique requires protein samples that are site-specifically equipped with a pair of donor and acceptor fluorophores. Here, we present a detailed protocol for preparing double-labeled proteins for smFRET ...
    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: 6782, 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: 8882, 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 ...

    We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.