I'm overexpressing a gene in a fibroblast cell line. I also over-express it via transfection in 293T as a positive control.
After stably transducing the cell line, I detect the gene expression by both qPCR and western blot.
qPCR looks good that there is definitely much more RNA level than the control.
However, for the western blot, there is no band on my over-expressed cell line. But there is a strong band for my positive control.
The difference between my positive control and the over-expressed cell line is that 1. 293T vs. fibroblast; 2. transfection vs. virus transduction
Why is there RNA detected in my OE cells but the protein is not detected?
Hi, Consider following questions for trouble shooting,
What about the reading frame of the gene after cloning into viral expression vector?
Did you check the protein over expression after transient transfection?
Is any induction is required(like Tet-on/off system)?
Have you used any positive control of your protein in western blotting?
All the best.
Hi, Suipwksiow.
It is also possible that the viral insertion occurs in a vulnerable site where a non-coding RNA gene species is present in addition to the check-up points mentioned above.
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