Well, one possibility is that the tissues where it is normally expressed also express a chaperone that move it to the ER, or do NOT express a chaperone that moves it to the golgi. Another is that there's some post-translational modification that only happens in the normal tissues. Another is that by transfection you're essentially overloading the cell, so you see a lot of localization in other places.
I'd try getting a brain or lung cell line, transfect into that, and see where the protein localizes.
Well, one possibility is that the tissues where it is normally expressed also express a chaperone that move it to the ER, or do NOT express a chaperone that moves it to the golgi. Another is that there's some post-translational modification that only happens in the normal tissues. Another is that by transfection you're essentially overloading the cell, so you see a lot of localization in other places.
I'd try getting a brain or lung cell line, transfect into that, and see where the protein localizes.
Hi there!
Before I suggest anything, what about the localization of your protein when you transiently overexpress it? Does it still go to ER or not? Have you checked the sequence of your clone? It might be some mutations in the signal sequences leading to improper localisation, because in both the cell lines it is going to golgi instead of ER. Check if your protein is always in golgi from the very start of its transient expression. Also, make sure your protein is properly folded to expose the right localization signals needed to transport it in required compartment. Is the protein active? Can you check that?
Hello Jia,
Another think to consider is the level of expression. If you over-express a protein too high, its trafficking will be affected. Based on what you said, I am pretty sure that you will find some in your culture supernatant. Try to lower the expression of your clone by driving gene expression with another promoter or an inducible and rheostatic system.
Hope that is of help
Cheers
Please, take into consideration that a DNA vector, a transfection reagent, expression of an antibiotic resistance (trans)gene, expression of a reporter (trans)gene, and selection by acute/chronic antibiotic treatment may evoke cellular responses that affect the biochemical processes under investigation. In the following review, we demonstrate that an assumption that empty vector-transfected cells preserve the cytogenetic and phenotypic characteristics, and represent the adequate control in transfection experiments is not universally valid. We exemplify a number of studies, which reported obvious genomic, transcriptomic and phenotypic changes of tumor cells after transient/stable transfection of an empty vector. Finally, we conceptualize that the diverse experimental manipulations (e.g., transgene overexpression, gene knock out/down, chemical treatments, acute changes in culture conditions, etc.) may act as a system stress, promoting intensive genome-level alterations (chromosomal instability, CIN), epigenetic and phenotypic alterations, which are beyond the function of manipulated genes.
Please see the following manuscript for details:
Stepanenko AA, Heng HH. Transient and stable vector transfection: Pitfalls, off-target effects, artifacts. Mutat Res. 2017 Jul;773:91-103. doi: 10.1016/j.mrrev.2017.05.002.
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