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

Published: May 31, 2023

Abstract

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 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.

Protocol

1. Preparation of agarose pad (Figure 1) 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. Pipette and pour a droplet of 70 µL of 1% melted agarose on the glass slide. Add a fourth slide on the top to flatten the ag…

Representative Results

Figure 1.  Agarose pad preparation. Figure 2. Schematic representation of the interface of microscope control software

Açıklamalar

The authors have nothing to disclose.

Materials

525/50 nm band-pass filter F37-516, AHF, Germany
680 nm short pass filter F37-516, AHF, Germany
Agarose Sigma-Aldrich  A9539
Fiber-coupled avalanche photo-diode SPCM-AQR-14- FC, Perkin Elmer
Glass coverslips (Thickness No. 1.5, 20×20mm Knitel glass MS0011
Microscope slides (25×75mm) Knitel glass MS0057
TCSPC module SPC830, Becker & Hickl, Germany
Ti:Sapphire laser Insight DeepSee, Spectra Physics

Etiketler

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Bu Makaleden Alıntı Yapın
FLIM-FRET Imaging for Characterization of Protein-Protein Interactions in Live Bacteria. J. Vis. Exp. (Pending Publication), e21394, doi: (2023).

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