Hi everyone. I have been trying to express my fusion protein to the outside membrane of the cell. My fusion protein contains eGFP. After lentivirus transduction, I observed a membrane-bound phenotype but the GFP signals were very low. Even after antibiotic selection, the signals did not improve. When I did western blot, probing for GFP and V5 tag, both of them showed high expressions.
Has anyone encountered this same dilemma?
How were you able to increase GFP expression?
TIA!
ehy Bryan, Can it be that maybe the fluorescent microscope settings are not the best for the wave of lenght that you are looking at?
Have you try to check if you are in the right range of wave lenght? it might seems silly, but sometimes it can happen. it seems strange also to me that you have low singal even if in WB you have high expression, therefore I was thinking at some kind of "error" in visualization , but not in the technique or in the byological aspect.
cheers
Federico
Hi Bryan, I second Jose's answer. External acidic pH is reduces a lot the fluorescence of eGFP. ( https://www.caister.com/backlist/mbt/v/v2/01.pdf)
Maybe you should try fusing your protein to "pH stable" versions of GFP. (i.e. https://www.nature.com/articles/srep28166). This one is available from Addgene (https://www.addgene.org/browse/article/18758/)
Cheers,
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