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Dear live-cell lovers,
I'm starting a project on protein trafficking in hippocampal neurons in
culture. I'd need several probes to localize proteins of interest
respective to organelles and cytoskeleton, and I'd be very happy to have
advices about which probe to use. Firtst, the setup is optimized for use
of eGFP and mCherry fluorescent proteins, so probes that fall in these
spectra would be great, but I can get additional filters (although the
Sutter DG-4 illumination system I have allows me to only use 3
excitation filters max.)
1) I'd like to detect cell compartments such as plasma membrane, Golgi,
ER. The first possibility is to express a targeted fluorescent protein,
such as those sold by Clontech some time ago, or the new Organelle Light
system from Invitrogen/Molecular Probes (but will baculovirus infect and
express proteins in rat hippocampal neurones in culture ?). If someone
has any old Clontech probe plasmid in the fridge (membrane, ER, Golgi)
I'd be happy to discuss about how to use them. The other possibility are
lipids or synthetic probes like dextran, I don't know a lot about
specific probes for the ER/Golgi/membrane that would fit in terms of
spectrum (ideally distinct from eGFP to monitor my protein of interest
in parallel) and work well in neurons.
2) I'd like to monitor actin and microtubules. Are there fluorescent
proteins available, either from a lab or from a company, that will allow
to visalize the cytoskeleton with minimal perturbation ? mCherry-actin
would be great. For microtubules, I've seen Oregon Green Taxol from
Molecular Probes, but I'm wondering what is the extent of the
perturbation induced to microtubules.
In brief, I'd like to see the ER, Golgi or plasma membrane, the actin or
microtubule cytoskeleton, in live cells, either with fluorescent protein
or synthetic probes, ideally with a spectrum similar to mCherry (max ex.
587 nm, em 610 nm), or similar to eGFP.
Thanks a lot !
--
Christophe Leterrier
Postdoc
INSERM UMR641 Neurobiology of ionic channels (Michael Seagar)
Ionic channels and neuronal polarity team (Bénédicte Dargent)
IFR Jean Roche - Mediterranee University
51, bd Pierre Dramard 13916 Marseille cedex 20 France
/http://ifrjr.nord.univ-mrs.fr/-Institut-Jean-Roche-?lang=en/
tel: (33) 4 91 69 89 33
fax: (33) 4 91 09 05 06
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