E. coli cells expressing either HdeA-wt or HdeA-F28KStyr were collected by centrifugation (21000 g) for 5 min at 4 °C. The cell pellets were washed three times with PBS buffer and re-suspended in PBS buffer containing 5% glycerol. Tz stock solution was added to give a final concentration of 100 μM. After 1 hour of incubation, 10 μL of the cell suspension was placed on top of a glass slip and covered by a glass cover slip. The glass slip-cell-slip sandwich was irradiated at 302 nm using a handheld UV lamp for 60 s. Cells were immediately imaged using Olympus FV500 inverted (Olympus IX-81) confocal microscope. Cells were excited with DAPI excitation wavelength (405 nm) and imaged using GFP emission filter (510nm).
Fv500 inverted 9 81 confocal microscope
Manufactured by Olympus
The FV500 inverted (Olympus IX-81) confocal microscope is a laboratory instrument designed for high-resolution imaging. It features an inverted optical configuration and a confocal scanning system to enable optical sectioning and three-dimensional imaging of samples.
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Lab products found in correlation
2 protocols using fv500 inverted 9 81 confocal microscope
1
Photocrosslinking of E. coli Cells
2
Bimolecular Fluorescence Complementation Assay
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The BiFC assay based on split YFP was used to test the interaction of E2 proteins and SlPUB13 in N. benthamiana leaf protoplasts (Chen et al., 2006 (link); Waadt et al., 2008 (link); Zhou et al., 2017 (link)). The non-group III E2 UBC13 fused to the N-terminus of YFP and the empty vectors expressing the N-terminus and C-terminus of YFP (nYFP-EV and cYFP-EV) were used as negative control. Protoplasts were prepared from leaves of wild type N. benthamiana plants as described (Rosebrock et al., 2007 (link)). Approximately 1 × 104 protoplasts that were suspended in a volume of 200 μL were then co-transfected with 10 μg plasmid DNA of each individual of the construct pair to be tested. The co-transfected protoplast was imaged 21 h after transfection using an Olympus FV500 Inverted (Olympus IX-81) Confocal Microscope with the following excitation (ex) and emission (em) wavelengths: YFP, 514.5 nm (ex), 525–555 nm (em); chlorophyll auto-fluorescence, 640.5 nm (ex) and 663–738 nm (em).
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