I had this crazy idea that I could use a GFP-fusion protein to measure
expression levels of a particular protein. There are no good antibodies to
this protein. We have been doing a lot of transient transfections of
wild-type and mutant protein and examining activity. We need to show that
the mutants have equal expression to the wildtype. I transfected the
plasmids and looked under the scope and saw a good deal of fluorescence in
the fusion protein-GFP cells but not the sham transfected (no GFP).
However, when I brought the cells to the fluorescence plate reader, I saw
no difference between sham and GFP cells. I went ahead and lysed the cells
for a reporter assay and came back to measure fluroescence again and the
values did not change.
Here are my questions:
Can GFP expression be quantitated by fluorescence spectroscopy?
Do GFPs fluoresce in solution (i.e. in lysed extracts)?
Any idea about the senstivity of spectroscopy versus doing western blots
with anti-GFP antibodies?
Thanks for your help
Jack Vanden Heuvel
104 Henning Bldg.
Department of Veterinary Science
Penn State University
University Park, PA 16802
jpv2 at psu.edu