I have two plasmids on the same backbone. Both of them have eGFP. The only difference is that one plasmid has 3x flag before the insert and the other doesn't have. When I transfect HEK293T cells using Lipofectamine LTX, I noticed eGFP is brighter in the plasmid without 3x flag. Does this make sense? Will 3x flag make eGFP expression less bright?
Dear Abdalla,
The transfection success depends of the plasmid length and the intra protein interaction. Maybe the three flags are became the plasmid so big to cross the nuclear pore or changing the tertiary protein structure (and consequently carrying to a post-translation degradation). I am not sure about the flag function (is like a tail for a semi-purification?!) but probably it is the cause of low transfection. Is the GFP just a reporter or the only protein that you are transfecting? If is GFP just a reporter, you could check the protein level before to discard the plasmid. If the GFP is the only protein and the plasmid is so important, you could change the agent transfection (I tried Lipofectamine 2000, calcium phosphate, Effectene and FuGene in HEK293T cells and the FuGene was the best for transient transfection). I change the cells source too (the same lineage but from different lab) and my production increase 30%!
Good luck!
Hi Tais,
Thanks for your answer. The GFP is just a reporter to confirm the transfection. The issue is the cells are GFP positive and the transfection efficiency is high but the brightness is weak compared to the other gene on the same backbone without the flag. The good news, the protein expression is as expected. I just wanted to understand why?
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