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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:
2748,
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:
3783,
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:
6466,
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:
8564,
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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