Difference between revisions of "Team:TU-Eindhoven/Demonstrate"
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<figcaption>Figure 1: Fluorescence measurements, FRET indicates close proximity</figcaption></div></br></br> | <figcaption>Figure 1: Fluorescence measurements, FRET indicates close proximity</figcaption></div></br></br> | ||
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Additionally, we can even visualize large clusters by measuring one of the fluorophores and compare this with all the other states described above. </br></br> | Additionally, we can even visualize large clusters by measuring one of the fluorophores and compare this with all the other states described above. </br></br> | ||
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Revision as of 18:26, 1 November 2017
Overview
Below you can find a description about the approaches that we used to proof that we indeed have made two constructs that will aggregate and form a large structure, similar to a gel.
We will investigate the following compositions:
- Scaffold Construct (14-3-3 with GFP)
- Binding Partner (CT33 with Strep-tag and mCherry)
- Inducer added to all needed constructs (14-3-3 with GFP, CT33 with Strep-tag and mCherry, Strep-tactin_
- No Inducer but with al needed constructs
- Inducer and all constructs needed, except Strep-tactin
- No Inducer and no Strep-tactin, with all the other constructs
FRET Measurements
The first approach is done with a plate reader measurement. The first step is to excite the fluorophore GFP, which will then emit light with a higher wavelength. A part of this emitted light has a wavelength that can excite the fluorophore mCherry, which will then also emit light with an even higher wavelength. This principle is called Föster Resonance Energy Transfer (FRET)[1] and mostly used to indicate a close proximity between two proteins.
In the case that our system doesn't behave as expected, there would be no difference between the induced and non-induced state.
Figure 1: Fluorescence measurements, FRET indicates close proximity
Microscopy
An even better indication of close proximity between the two constructs can be generated by using a microscope. With the microscope we can make two pictures, one where we visualize GFP and one where we visualize mCherry, and overlay them to see that the two different constructs are close to each other.
Additionally, we can even visualize large clusters by measuring one of the fluorophores and compare this with all the other states described above.
Figure 2: Fluorescence microscopy images after 48 h of incubation. Signal from samples with all GUPPI components (A, B and C) and samples without streptactin (D, E and F), leaving the CT33 protein with a single valency. A) GFP signal B) mCherry signal C) signal overlay D) GFP signal E) mCherry signal F) signal overlay. Data was modified using ImageJ
[1] B.T. Bajar, E.S. Wang, S. Zhang, M.Z. Lin and J. Chu, "A Guide to Fluorescent Protein FRET Pairs", Sensors (Basel), vol 16, no. 9, pp 1488, (2016)
