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GFP-tagged GPI-anchored protein expression

GPI egfp cloning

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#1 Keller

Keller

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Posted 05 September 2011 - 12:56 AM

I need to tag a GPI-anchored protein to see its expression and incorporation into the plasma membrane. By logic I should add it after the signal peptide, but when I treat with PI-PLC I would lose the signal and would not be able to know if a particular cell is transfected or not. So for the sake of it, I added EGFP to the very C-terminus of the protein, meaning even after the region that is removed for anchorage. Interestingly, I see a GFP signal along the membrane, which means it has been correctly incorporated to the plasma membrane and is not stuck somewhere in the ER. Is there any explanation for this? And why do I see the signal even after PI-PLC treatment? I very confused, and would greatly appreciate your help.





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