Regarding Marty's question on spectral shifts of fluorescent proteins, this
is an important concern and it would be nice if a Clontech representative
jumps on and gives us an answer, since they generate and market these
constructs.
I think Kevin's compensation concern is an important one when fluorescent
proteins are involved in the mix, since the intensity can be quite bright
and span a few decades. With this type of fluorescence intensity
distribution, single-color compensated parameters can look strange, and I
think this may be due to slightly nonlinear amplifiers on older systems.
FRET with FPs can be frustrating in flow (and more so in microscopy), and
I've found proper compensation to be critical for my flow setup. It's
important to use single color positive controls as usual, but don't base
your instrument setup on settings from an untransfected cell line, FP
positive cells of a different cell type, or FP positive cells where the
linked protein isn't the one of interest. I found the gains, compensation,
laser power, etc. will not work unless you use the actual transfected cell
expressing the single-color FP of interest as a positive control.
Lastly, if you have a system that can store uncompensated data , you can
tweak in the compensation offline, assuming the optical and detector
settings were correct.
These references may be helpful:
PNAS 2002; 99: 7102-7107.
J. Biol. Chem. 2002;277(5):3767-75
Peter Lopez
The Aaron Diamond AIDS Research Center
212.448.5188 (office)
212.448.5158 (fax)
212.448.5190 or 5110 (lab)
Kevin Holmes
<KHOLMES@niaid.ni To: Cytometry Mailing List
h.gov> <cytometry@flowcyt.cyto.purdue.edu>
cc:
08/13/2002 04:45 Subject: RE: gfp spectral shifts?
PM
Marty,
Interesting question. When we have performed FRET experiments using YFP
and CFP, we've noticed that when performing compensation between CFP and
the detector used to collect FRET emission from YFP, it can be difficult to
correctly compensate both the bright and dull CFP+ cells. That is, if the
bright +'s were correctly compensated, the dull+'s were overcompensated;
if the dull+'s were correctly compensated the bright+'s were
undercompensated. This effect was seen in CFP+ only cells. My thought has
been that there is a change in the emission spectrum of CFP dependent upon
concentration and/or some kind of spectral quenching effect occurring.
Unfortunately, I don't have any firm data to confirm the above hypothesis.
Kevin
Kevin L. Holmes, Ph.D.
Chief, Flow Cytometry Section
Research Technologies Branch
Bldg. 4, Room B1-38
NIAID, NIH
Phone: 301-496-9071
FAX: 301-402-4532
Email: kholmes@niaid.nih.gov
-----Original Message-----
From: Marty Bigos [mailto:mbigos@gladstone.ucsf.edu]
Sent: Monday, August 12, 2002 10:47 AM
To: cyto-inbox
Subject: gfp spectral shifts?
Hi -
Does anyone know of shifts in the emission spectra of the various gfp
constructs due to intracellular pH, linkage to other proteins, etc.?
Thanks.
Marty
--
Marty Bigos
Director, Flow Core
Gladstone Institute of Virology and Immunology
Building 3 SFGH Rm 509
415-695-3832
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