dsRFP detection

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Thu Feb 17 04:41:16 EST 2000


Jered Floyd wrote:

> MMILLING at MEDAU.JNJ.com ("MILLINGTON, Michelle [JJRAU]") writes:
> > We have tried EBFP but are unable to detect by flow due to  low fluorescence
> > and photobleaching.  Is dsRFP brighter and more resistant to photobleaching
> > than EBFP?
>
> With an epifluorescence microscope, DsRed is significantly brighter
> and much more resistant to photobleaching than GFP/EGFP;

We´ve also used RFP and EGFP, but with reverse result: EGFP is MUCH brighter the
RFP, at least in COS cells transfected with vectors dsRed-N1 and pEGFP-N1,
respectively.

> I imagine as
> such it is much better than EBFP as well.  I have not yet tried it
> with flow, but will soon.
>
> One word of warning with DsRed: At least in E. coli, there is a very
> large delay (36-48 hours) before it is visible to the eye. This is
> unfortunate, as the other FPs express visibly much faster.
>

Not only in E.coli. When looking at transfected cells, RFP is not detectable 24h
after transfection (when EGFP is already VERY bright), but shows a good signal
only after at least 48h. Seems the "maturation" of the protein - which makes it
fluorescent - takes much longer for RFP than for GFP. This is an important thing
to remember when doing cotransfections with RFP and GFP.

Frank







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