I am expressing a recombinant mcherry red protein by using a pVAC1-mcs vector in HEK293 cells. pVAC1-mcs has a 3' glycosylphosphatidylinositol (GPI) anchoring domain of placental alkaline phosphatase (PLAP). Based on that, the recombinant mcherry red protein should be on the membrane with the GPI anchor. I have results supporting the presence of mcherry red (fluorescent micrographs, membrane protein western blots) but the phospholipase C mediated release of mcherry red protein is not working. Does anyone have experience with this?
Hello,
Did you use PI-PLC by adding directly to cells? I have no experience on that, but is described elsewhere that it should work (check concnetrations just in case). You could also try to get membranes, and then perform the two-phase separation by Triton-X114 in combination with PI-PLC. This approach has been proved to work with many GPI-anchored proteins.You could also try with phospholipase D (PLD), although I am not aware of highly purified PLD on marked (I worked with it in a proteomics context, and purity of available ones was not good enough)
Good luck!
Felix Elortza
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