Hi all!
I want to use acceptor photobleaching FRET to look at a potential interaction between two proteins of interest. We have a Leica SP8 in my lab that I have used to try and get an idea if this would be possible.
I have three sets of coverslips with fixed HeLa cells transfected with the following:
- Protein A(GFP) + Protein A(mCherry) (dimerises so positive control)
- Protein A(GFP) + Protein B(mCherry) (definitely doesn't dimerises so neg control)
- Protein A(GFP) + Protein C(mCherry) protein of interest so let's find out!
For each of these, I have taken an in both channels, then bleached the red channel and re-imaged both channels. I draw ROIs and measure mean grey levels to quantify fluorescence before and after bleaching and calculate the change in fluorescence and the fret efficiency.
However, my positive and negative controls have similar values.
Nobody in my lab does AP FRET so I'm worried I'm missing out a step in the experiment or the analysis and have nobody to help. Can anybody advise me on ways I can improve this set up?
Is your bleaching effective? I want to see at least 80% reduction in acceptor intensity in the ROI. I'm not sure how good your positive control is because FRET depends a lot on things like distance and dipole orientation of the fluorophores. Depending on the actual arrangement of this construct you may not get FRET.
In my experience if you have good photobleaching of the acceptor you will see a visible, clear increase in donor fluorescence in the same ROI if there is FRET.
Keep the detector gain low if you can. Noise can potentially bury small intensity changes if FRET is weak.
Hi Becky,
I don't know if you are still having trouble with your acceptor photobleaching FRET. Although this is simple approach as far as the theory is concerned, it has some pitfalls. You should check the following:
- how much reduction in the acceptor intensity do you get after bleaching? (as also asked by Christopher)
- do you get a change in the donor intensity if you bleach a donor-only sample at the acceptor wavelength?
- how much overspill do you have from the acceptor to the donor channel?
- do you get a change in the donor channel if you photobleach an acceptor-only sample?
You could try our ImageJ plugin available here:
https://biophys.med.unideb.hu/en/accpbfret
based on the following publication:
Article AccPbFRET: An ImageJ plugin for semi-automatic, fully correc...
It describes all the necessary controls for acceptor photobleaching FRET.
Good luck,
Peter
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence