I have a cell line that expresses an endogenous protein. Now, if I transfect overexpression in the same protein, how do I know that the results I am getting are due to the overexpression protein?
You should always compare your results to a mock transfection (with an empty vector or a vector with a different unrelated protein). If the mock transfection is significantly different than your overexpression your results are due to the overexpression.
Hi, Amruta, do you mean protein or mRNA level?
If you measure the protein, you can isolated rptein form control and transgenic lines parallel, load to the gel exactly the same amount of protein and probe with specific antibody. Differences between control and transgenic (over expression) lines will give you expression level. If you protein is the same as natural, it will be very hard to distinguish between natural and additional one. Good luck!
You should always compare your results to a mock transfection (with an empty vector or a vector with a different unrelated protein). If the mock transfection is significantly different than your overexpression your results are due to the overexpression.
I am thinking of adding a myc tag to my insert. I am using a conditional cell line where overexpression of my protein is only seen under stress. I would put stress on cells with a drug that would trigger overexpression. Then conduct a western blot with cell lysates. The tagged vector will generate significant amount of protein as compared to endogenous expression. It will generate separate bands on WB, aiding to distinguish between them. Please someone let me know if this sounds correct.
Hi, Amruta, it looks reasonable, you can load on the the WB samples before and after treatments and compare with MYC antibody. However, you definitely need an additional control, ea. to check how native protein level alter after treatments! On the separate membranes with the Ab against native protein. Good luck!
Hi, Ahed, I agree with you, immunolocalization can be consider as alternative to WB to study protein level, and one can make a probe against MYC to see recombinant protein level upon stimulation. But it is highly dependent from cell types you have used..
You usually tag your protein, so it has different size or color or both
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