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 dear listserve members,

I have done my first FRET Sensitized Emission
experiment using Leica TCS SP2 CLSM and would like to
request you all to give your valuable suggestions. The
FRET pairs are: donor,  EGFP and Acceptor, Alexa 546.
I have done live cell imaging which means that the
cells were not fixed but certainly washed in PBS,
thrice. I have used 488nm LASER line for donor
excitation and 543nm for acceptor excitation.I have
followed the guidelines of the FRET SE version 1537
protocol , but have certain queries to make.

I must also mention that I am trying to study Protein
and RNA interaction for which I have tagged the
protein with EGFP and labeled the RNA with Alexa546.
This protein interacts with RNA and has been shown by
us by other experiments as well. But we want to prove
the same with FRET. Along with the FRET references
i.e. only DONOR (protein tagged to EGFP), only
Acceptor (Alexa546 labeled RNA) and FRET sample
(having both of these)i have also included another
FRET sample only EGFP and Alexa546 labeled RNA. Only
EGFP should not interact with Alexa546 labeled RNA
after all this is why it is used as a reporter.

I have done offline quantification using the FRET SE
software.I expected  higher FRET efficiency incase of
protein tagged to EGFP and Alexa546 labeled RNA. But i
am getting similar values with only EGFP and Alexa546
labeled RNA, which probably should not happen. I have
taken approximately 50-70 ROIs in each case. The
values of FRET efficiency are as high as 50, 30, 25
and at some ROIs even above 100, in both of these
cases.

I request you all to give your suggestion which would
be of immense help to me.

Thanking you,
Shagufta Rehman












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