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Using FRET to show formation of homo-dimers. No signal?! Input appreciated&#

FRET dimerization

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#1 sddk

sddk

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Posted 15 August 2013 - 07:29 AM

Hi!
I'm new here and I would love to get some input on the problems I face with FRET. I'm coming from a pure biology background and I don't really have anyone here experienced enough to help me with anything FRET-related. I am supposed to set up FRET experiments in our lab and I'm sure I missed tons of stuff that is probably obvious for microscopy people.


In short, I'm trying to measure FRET between the two monomers of a transmembrane receptor.

Little Background:
Upon activation by the ligand, the two RTK monomers are supposed to dimerize which leads to auto-Phosphorylation of the intracellular part of each monomer -> signal cascade etc.
Now, as it was not actually shown yet for this RTK, I want to show this dimerization itself. Actually, the end goal would be, to test if other proteins are possibly involved in the dimer formation (e.g. co-receptors that were shown to be required for the activation of this receptor).

Protocol related:
For all my vectors, I fused the respective fluorophore to the c-terminal end of the RTK (=the intracellular part). I tried several different linker sequences in between them.

I started out with CFP&EYFP as a FRET pair, tried also EYFP&mCherry and now I'm mainly using mEGFP&mRFP1. The reason being that these GFP&RPF are both monomeric and don't already dimerize by themselves (although I now read this is a rather weak artifact to worry about?).

So for the experiment, I transiently co-transfect RTK-GFP and RTK-RFP into 293T cells seeded on glass slides
The cells are starved and then induced with the ligand of the RTK for 5 minutes (For Western Blot, this is enough to see the activation of the signaling cascade, so any dimers should have been formed).
I fix them with 4% PFA for ca. 10 minutes at RT, wash 3 times with PBS and cover them with fluorescent mounting medium (Dako). A day+ later, I check them under our confocal microscope.

Measured at the same time with 488 nm laser:
Channel 1, GFP (495nm - 545nm) Gain: 760
Channel 2, FRET (590nm - 660nm) Active, Gain: 710

Measured with 561 nm laser:
Channel 2, RFP (590nm - 660nm) Active, Gain: 710

I try to equalize the signal strengths of GFP and RFP. The RFP signal is very weak, so the 561 laser is set much higher than the 488 laser.


Problems:
My main problem is that I don't seem to have a difference between the noninduced (should be no dimer = no FRET) and induced condition (dimer = FRET). As I'm not experienced in FRET, I don't really know what to expect. I was under the assumption, that I should already see a difference in the raw FRET channel (GFP excitation, RFP emission). After bleed-through correction and acceptor-normalization (I'm using a imageJ FRET plugin), some cells may show a difference. Problem for statistics is, that the histograms of the different conditions are still so close together.
I tried some acceptor photobleaching, but didn't see an increase of GFP signal.

Also:
- Is the GFP/RFP pair even suitable for my FRET experiments? I read it's more for FLIM-FRET stuff, not for sensitized emission or acceptor bleaching FRET. But I don't quite get why.
- Would it make sense to clone and screen fusion protein constructs with different linkers between the RTK and the GFP/RPF to see if one gives me better FRET? I already tried 5 between 5 and 20 amino acids long linkers with different flexibility.
- Is it likely due to the settings of the microscope? It's not so easy to reduce GFP bleed-through into the FRET channel and still get a sufficient RFP signal with the same gain/filters etc,

 

 

(I hope this is the right forum, otherwise please point me in the right direction)

I'm sorry if this is too long, but I wanted to include most of the details. Any help is appreciated.
Cheers,
David

 






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