Antibody binding to nonpermeabilized cells

Dr Engelbert Buxbaum via methods%40net.bio.net (by engelbert_buxbaum from hotmail.com)
Thu Nov 15 08:40:48 EST 2007


Am 06.11.2007, 15:06 Uhr, schrieb Beug, Shawn <sbeug from ohri.ca>:

> I am interested in calculating the ratio of intracellular and
> extracellular GFP-tagged protein in primary neurons. I am trying to
> demonstrate that perturbation of posttranslational modification affects
> extracellular/intracellular sublocalization. I was wondering if
> immunnostaining for transfected neurons with an anti-GFP antibody

Could be done. The easier way is to work with a non-membranepermeable  
fluorescence quencher (iodide, acrylamide or the like). The fluorescence  
of extracellular GFP would be quenched, that of intracellular GFP not, the  
ratio of fluorescence with/without quencher is, under appropriate  
conditions, equal to the ratio of intracellular / total GFP.

See:

@book{Lak-99,
         AUTHOR= {J.R. Lakowicz},
         TITLE= {Principles of fluorescence spectroscopy},
         YEAR= {1999},
         PUBLISHER= {Kluwer},
         ADDRESS= {New York},
         EDITION= {Second},
         LANGUAGE= {engl}
}

p.s.: It makes little sense to add a several language confidentiality  
statement to a message published on usenet.


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