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
Disclosures
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 |
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Cite This Article
FLIM-FRET Imaging for Characterization of Protein-Protein Interactions in Live Bacteria. J. Vis. Exp. (Pending Publication), e21394, doi: (2023).