identification of the transfection efficiency - (Sep/18/2011 )
The methods of confirm the stable transfection is successful or not is add antibiotic to kill untransfected cell, carry out the Western Blot for detect the expressed protein, run PCR to detect the gene inside the cell, and using green fluorescent protein gene as control.
Do anyone know the other methods to confirm the cell is successfully transfected and stable (continue) express the desired protein?
Thank you very much.
Homework buy the sounds of it...
Why would you use GFP? Why wouldn't you use an antibody against the protein itself?
Thank you for your reply, GFP gene was come together with transfection reagent. I was used antibody against the protein in WB, but I couldn't get the desired protein band show in membrane. So I think the gene is not transfect into cell. Then, I run PCR as normal but the result is negative.
qPCR is for quantitative, I just want to know my gene is transfected or not. Do you know any detection methods after transfection?
Detection by western blotting is the end-point measure of whether you have transfected the cells, and as such is a good technique to use, as you are likely to use it for many different experiments in the future. You can use immunofluorescence or immunohistochemistry to see if you have a low transfection efficiency (i.e. some cells transfect, but others don't), it is more sensitive than westerns, as you can see the individual cells, but it doesn't tell you the same things. You should work on optimising these!
Adding antibiotics does not tell you if the cells are transfected unless you have very good controls, some cells may survive even if they are untransfected. You would still need to prove that your cells were expressing the protein.
qPCR allows you to detect low levels of genes in the total cell population - if you have poor transfection efficiency, you may be able to detect some signal with qPCR, that will tell you if you transfected at a low level, or if there is something wrong with the plasmid.
Thank you very much