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1. Preparation of agarose pad (Figure 1)
<ol>
	<li>Place a microscope glass slide on a flat horizontal surface. Arrange two glass slides topped with two layers of adhesive tape on each side of the initial slide. 
	NOTE: Keep a 1-2 mm space between the three aligned slides to prevent the melted agarose from eventually spreading on the slides with adhesive tape.</li>
	<li>Pipette and pour a droplet of 70 &#181;L of 1% melted agarose on the glass slide. Add a fourth slide on the top to flatten the ag...

flim-fret imaging for characterization of protein-protein interactions in live bacteria

Source: Manko, H. et al., <a href="https://app.jove.com/v/61602">FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.</a> J. Vis. Exp. (2020)
The video describes the FLIM-FRET imaging technique to determine the protein-protein interaction in live bacteria expressing cytoplasmic proteins labeled with fluorescent proteins, a donor eGFP, and acceptor mCherry. The combined technique also allows the quantification of the interacting proteins.

<img alt="Figure 1" src="/files/ftp_upload/21394/21394_Figure1.jpg" /> 
Figure 1.&#160; Agarose pad preparation.
<img alt="Figure 2" src="/files/ftp_upload/21394/21394_Figure2.jpg" /> 
Figure 2. Schematic representation of the interface of microscope control software

FLIM-FRET Imaging for Characterization of Protein-Protein Interactions in Live Bacteria

Source: Manko, H. et al., FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria. J. Vis. Exp. (2020)
The video describes the FLIM-FRET ...

To study specific protein-protein interactions in live bacteria using fluorescence lifetime imaging microscopy, FLIM, combined with F&#246;rster resonance energy transfer, FRET, begin with a recombinant bacterial suspension. The bacteria express specific cytoplasmic proteins involved in metabolic pathways, labeled with donor and acceptor fluorescent proteins suitable for FRET.
Spot the bacterial suspension on an agarose pad, which immobilizes the bacteria on a microscope slide. Place a coverslip on the slide and seal.
Position the slide on the stage of a two-photon excitation inverted scanning microscope for FLIM-FRET microscopy. Use a fluorescence lamp to select and scan a region of interest on the bacterial monolayer.
The interaction between the expressed cytoplasmic proteins brings the donor and acceptor fluorescent proteins closer in appropriate orientation. Upon laser illumination with a suitable wavelength, the donor fluorescent protein gets excited.
The close proximity of fluorescent proteins triggers FRET, leading to non-radiative energy transfer from donor to acceptor fluorescent proteins, which gets excited and eventually returns to the ground state, emitting fluorescence.
The non-radiative energy transfer reduces the time taken for the excited donor fluorescent protein to return to its ground state, the donor fluorescence lifetime &#8212; measured by FLIM. The decrease in the donor fluorescent protein&#39;s mean fluorescence lifetime owing to FRET confirms the protein-protein interaction in the bacteria.

flim-fret-imaging-for-characterization-protein-protein-interactions

Research

JoVE Encyclopedia of Experiments

Biological Techniques

Biomolecular Interaction Detection Techniques

Protein-protein interactions

The video describes the FLIM-FRET imaging technique to determine the protein-protein interaction in live bacteria expressing cytoplasmic proteins labeled with fluorescent proteins, a donor eGFP, and acceptor mCherry. The combined technique also allows the quantification of the interacting proteins.

Overview

Protocol

<img title="figure-representative results-58" alt="figure-representative results-58" src="/files/ftp_upload/21394/21394_Figure1.jpg" /> 
Figure 1.&#160; Agarose pad preparation.
<img title="figure-representative results-254" alt="figure-representative results-254" src="/files/ftp_upload/21394/21394_Figure2.jpg" /> 
Figure 2. Schematic representation of the interface of microscope control software

FLIM-FRET Imaging for Characterization of Protein-Protein Interactions in Live Bacteria

In This Article

Overview

Protocol

Representative Results

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