I need a protocol for the FRET analysis. - (Jan/08/2008 )
Hey guys, I was wondering if anyone of you has done the FRET analysis. I'm completely new to this technique and would appreciate any info/protocol/advice. Thanks!!!
Maggie.
Details please?
What fluorophores will you use? What will you exactly be measuring by FRET? Do you have a confocal microscope, or will you be doing it by a microplate reader or maybe a flow cytometer?
Thanks a lot for the reply!
Let me think...I'll be using GFP and DsRed. And I wanna measure interactions (heterodimerization) of the 2 membrane receptors. And yes, I do have the comfocal microscope. Oh, and I also can use the flow cytometer.
Let me think...I'll be using GFP and DsRed. And I wanna measure interactions (heterodimerization) of the 2 membrane receptors. And yes, I do have the comfocal microscope. Oh, and I also can use the flow cytometer.
your confocal microscope should be equipped with a software which may help to analyze FRET; if there is no special tool integrated, try imagej with appropriate plug-ins
I wonder if GFP and DsRed will work for FRET...
Might work, but I wouldn't try with DsRed as it's a tetramer. GFP-mRFP or GFP-mCherry are suitable, as are CFP-YFP.
Thanks! Now I was wondering...Do I have to have fusion proteins (my protein of interest fused with the fluorophore) or could I just use the secondary antibodies with teh fluorophores and in this way avoid the time-cloning step?
time-consuming cloning step, I ment 
Well...somebody just told me that one could also mark the primary Ab with fluorochrome and that it's pretty stright forward.
yes. Invitrogen/Molecular Probes sells ready to use kit for labeling.
Hi Maggie,
I have one FRET protocol from Tove if you want.