I cloned my GOI into a vector to add an N-terminal Flag tag and I am trying to verify expression after transient transfection by western blot with an anti-Flag M2 antibody (mouse mono) at 1:1000 dilution and 20ug total protein. My western does not show any any bands even after long exposures (10min). A major issue is that I do not have a positive control but I plan to get one. This construct is 10kb and GOI is 5kb. I have never transfected with such a large construct. Do I need to use more plasmid DNA compared to a construct that is 7-8kb? Either the transfection or western (i.e. antibody) is not working. I also tried a rabbit polyclonal anti-flag antibody at 1:1000 but got lots of bands. I do not care about detecting the mRNA in this case. I am trying to confirm expression of the Flag tagged protein.
Note: I transfected the same cell line exactly the same way but with another expression construct 7kb (HA tagged GOI - 2kb) and I was able to detect this protein with an anti-HA antibody. Ponceau S stain and actin ok for both.