Hi everyone
Background: Expressing a membrane protein in A549 cells (lung epithelial cancer cell line) using a expression vector under the constitutive CMV promoter. I am able to detect the expression of the protein 24hr post-transfection by immunofluorescence but I cannot detect the protein on a western blot, which I believe is due to low transfection efficiency (
Hi, it is possible that the CMV promoter is being methylated in your stable cells. You may find it useful to try a promoter such as EF1alpha. This promoter is less active than the CMV promoter so the cell is less likely to silence this promoter, at least it seems so in my past work. The reduced expression levels from this promoter also help with toxic proteins, which you mention you may be experiencing. Good luck.
Thank you Erik. I might actually give that a try as this was one of the things we wanted to try our luck with. If you are aware of any tightly controlled inducible system, let me know as well.
Well first things first, if you have a low transfection efficiency with your current protocol or method, then you must optimize or switch method. There is no point in trying to make a stable when you can't detect protein by western after transfection, since genomic integration is a random process, unless otherwise engineered, and since the locus of insertion has an influence on the expression levels, your chances of developing a good clone are lowered by low tranfection efficiency.
Using an inducible promoter system is typical how people have successfully dealt with toxic protein expression. I have attached a link with a paper that will give an overview of membrane protein over-expression in mamalian cells, transient and stable and examples of the expression of potentially toxic proteins using inducible systems.
Hope you find the literature useful.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3709882/
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