In reply to your questions, my target is an outer membrane protein located
on the surface of the organism and I am using live cells. I've had some luck
with that new conjugate I mentioned. It is called Alexa-fluor 488 and it's
made by Molecular Probes. The fluorophore is a FITC analog that excites at
488nm (which matches the frequency of the argon laser most cytometers use).
I've only done one experiment with it so far, but it dramatically reduced
the background in the negative control.
"Emir Khatipov" <khatipovNO at NOuchicago.edu> wrote in message
news:OEK%8.44$X4.10409 at news.uchicago.edu...
> I would also be interested to hear opinions on that subject. I myself try
> do similar things with mammalian cells, and ran out of ideas on how to
> reduce background. I guess in case of bacteria the problem should be even
> worse because of the cell wall or maybe other reasons. I also know that
> itself binds pretty strongly to membranes. However, it is obviously not
> case with fixed and permeabilized cells. Are you fixing the cells
> ((para)formaldehyde) before running FACS or work on live cells? What is
> target, surface or intracytoplasmic? If your Ab works in immunomicroscopy
> fixed cells, then it should work in FACS after minor protocol adjustments.
> - Emir
>> "John M Neary" <neary at acsu.buffalo.edu> wrote in message
> news:ahnnpu$r2p$1 at prometheus.acsu.buffalo.edu...> > Is there anyone on this board familiar with flow cytometry on indirectly
> > immunostained (primary Ab - labeled conjugate) bacteria? I'm getting
> > background and low signal with a FITC-labeled conjugate. I just
> > Alexa-488 labeled conjugate today. I heard good things, but haven't
> > it yet.