Transfection into a cell line which already express your protein - (Feb/17/2006 )
What does it happen when you express your protein (via transfection) within a cell line which already express it? Do you need to overexpreess your construct to make sure that it prevails over the native one? Is this convenient?
What happen if you don't overexpress them? To not overexpress how much mRNA should you use?
This is information which I find difficult to get from books or websites, so I thank your suggestions and ideas.
if you transfect a plasmid, selectable marker allows you to ensure the cell has your vector.
To see if protein is overexpressed, do a wester blot comparing non transfected cells to transfected ones (or transfected clones).
Thank you fred.
To assess the overexpression, I was thinking of using GFP tagged to my sequence, but that idea about Wester blot is good as well and might help me if I get problems with GFP.
How about the second part, do anyone work in transfection without actually overexpress their protein? In this case, it would be in a cell line with no expression of the protein of interest.
hello, nowadays im thinking about the same question, i think that maybe endogenous protein may interfere with the effect that you want to see....im thinking of considering siRNA....dont know how practical that would be...