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.
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).
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.
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.
- 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.