nh106 at cam.ac.uk
Wed Jan 17 14:36:55 EST 2007
In message <184.108.40.206.1.20070116102451.01c7e768 at unsw.edu.au>
Leonie Gaudry <L.gaudry at unsw.edu.au> wrote:
> I have been asked to do FRET using mCherry to GFP and also CFP to YFP.
> I was going to tune my second Laser to first 458nm for CFP then 568
> for the mCherry.
> Are there any alignment beads to confirm detection at these
> excitation wavelengths as wells as emission at 475nm for CFP anf
> 610nm for the mCherry?
> As we are looking for energy transfer I presume I would have to turn
> off the 488nm Laser. Does this mean triggering on fluorescence for
> the mCherry and CFP?
> I would appreciate any help from the community.
> Thank you
> Leonie Gaudry
I am going to ask several questions because I am not sure i follow
your plans. However, before that I will just summarise our experience
of doing CFP-YFP FRET on a MoFlo.
We used standard ECFP and EYFP protein fusions and excited at 407nm
for ECFY, FRET and 514nm for EFYP. We triggered on SSC (514nm).
We found this pair difficult as the contribution of ECFP only to the
FRET (546/10) channel was significant in relation to the weak FRET we
were detecting (as suggested by quenching of the ECFP (470/20)).
You can align with Spherotech Rainbow beads at all the wavelengths
within the desired range.
Since we did this work more optimal FRET pairs have been generated
(e.g. CyPet-YPet, see Nguyen and Daugherty Nature Biotechnology
23:355;2005). One non-ideal part of our setup was triggering off the
514 laser which meant we had to align that as first beam; in theroy
the lavel of light should not bleach the EYFP so not interfere with
subsequent FRET if any but it would have been nicer to trigger off the
donor excitation line and I suppose we could have done that if we had
triggered off fluorescence.
q1. I am not aware that you can get 458nm and 568nm from the same
laser. 568nm is a Krypton line which we have used for exciting mRFP.
q2. with GFP and mCherry, surely the GFP is the donor (I do not know
how effieicnt this ppair is), so surely you would want to excite at
488nm or similar to see FRET?
q3. what sort of instrument are you using? what lasers etc too?
Nick Holmes Division of Immunology, Dept of Pathology
Cambridge University, Cambridge CB2 1QP, UK
Tel:+44 1223 333871 Fax:+44 1223 333346
mailto:nh106 at cam.ac.uk http://www-immuno.path.cam.ac.uk/~nh106/
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