Hi, flowers,
We are beginning to do some experimentation with FRET and I have some
instrumentation questions. First, the experiment: E. coli has been
transfected with both GFP + proteinA and also with BFP+proteinB. Both
proteins are membrane-bound and don't come together unless a stimulus is
applied.
We have been using a 488 (argon) in the primary postion and a 405
(krypton) in the 2nd position to detect these two fluorophores on a
FACStarPlus. We have been looking at the GFP (530/30) alone off the 1st
laser and the BFP (460/20) off the second. In addition, we have a detector
that looks at any GFP generated by FRET off the second (also a
530/30--with a 505 beam splitter). Because the expression is low we have
been collecting signals with linear amplification--requiring increased gain
settings.
Question 1: Do we have the right instrument configuration to collect the
respective signals? The blue we see (when we can see it, is very dim).,
but the green is significant. (Stupid question --will exciting the GFP
with 488 first, render it unexcitable by the BFP if the beams are spatially
separated?)
Question 2: We are also collecting a ratio (GFP-second beam/BFP-second
beam) but have been having some difficulty because the blue signal has been
so dim. Is this an appropriate measurement?
Question 3: If the transfection efficiency of the BFP-proteinB combination
is bad, can I assume we are SOL? Should the signal intensity of both
fluorophores be somewhat equal to get good measurements?
Any suggestions would be greatly appreciated.
Thanks in advance.
Kathy
--------------------------------------------------------------
Kathy Schell
Supervisor, UWCCC Flow Cytometry Facility
600 Highland Ave. K4/535
Madison, WI 53792
Voice: 608-263-0313
e-mail: kschell@facstaff.wisc.edu
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