Hi Tony,

Type I proteins can be cleaved by proteases, which can account for your low signal. I placed an N-term FLAG-tag in a type I transmembrane protein to analyze this phenomenology (sorry not published). Check your growth media (or even starvation media) by western to see if the protein is being released from the membrane - you might need to concentrate the media before running the gel.

Best,

Joe


On 4/14/09 2:26 PM, "Anthony Kowal" <[log in to unmask]> wrote:


Hello All,

I was wondering if anyone knows of any (or has heard of any) plasma membrane proteins which have been tagged at the N-terminus with GFP.  I realize that the GFP coding sequence would have to be placed between the signal peptide sequence and the N-terminus of the protein, so that the fusion protein is targeted correctly to the secretory pathway.  I know of, and have used, Transmembrane proteins which have been tagged at the C-terminus with GFP, and both proteins are still functional.  Would one expect GFP to fluoresce extracellularly?

I have actually tried to tag a multipass transmembrane protein with GFP at the N-terminus (so it is extracullular), and although I observe GFP fluorescence, it is much weaker than when GFP is intracellular.  If I supplement the imaging media with ~20mM reduced glutathione, the fluorescence is more pronouced, but still weak.  The DNA sequence was verified, and all of the known amino acids for GFP to fold correctly and fluoresce are present.  The protein of interest is still functional.

Any thoughts/suggestions/ideas are very much appreciated.

Thank you for your time.

Best -

Tony


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Joseph Brzostowski, Ph.D.
LIG Imaging Facility, Head
National Institutes of Health/NIAID
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