I have made N-terminal fusions of EGFP to a hormone receptor
using CLONTECH's pEGFP-N1. I expressed the fusion protein in the
epithelial MDCK cell line. Just like you have observed,
detection of the fusion receptor was difficult using
conventional fluorescence microscopy. Since the protein was
membrane-targeted and only expressed in small amounts, only a
faint signal could be observed in the microscope. Westerns
showed that the MDCK cells expressed the fusion receptor at
significantly lower levels than the wt receptor in cells
transfected with the same amount of DNA.
We then turned towards confocal microscopy, which proved to be a
highly sensitive way of detecting the fusion receptor. Later, I
have been using FACS to analyze cells when the EGFP-signal
obtained has been to weak for fluorescence microscopy.
Jens Tornoe
NsGene, Denmark
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