identification of the transfection efficiencystable transfection
Posted 18 September 2011 - 08:06 AM
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.
Posted 18 September 2011 - 05:03 PM
Why would you use GFP? Why wouldn't you use an antibody against the protein itself?
Posted 18 September 2011 - 06:58 PM
qPCR is for quantitative, I just want to know my gene is transfected or not. Do you know any detection methods after transfection?
Posted 19 September 2011 - 05:31 PM
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.