Stable transfection (How to examine my control clones) - (Jul/10/2007 )
Dear All,
I am trying to clone my gene of interest into Hela cells for stable expression. My vector is 3*Flag-tagged and I transfected the empty vector as control. Now I am at the stage where I can lyse some colonies for sceening. The problem is if I would like to make sure the control vector (flag-tagged) is stably integrated into the cells and properly expresses at protein level, How I can do this. As even 3*Flag is about 8kDa, is it possible to check by western or is ELISA the best way to do it? Could anyone give some suggestion? Great thanks would be given.
Thanks,
Nan 
Hi Nan,
I tried to detect 3xFLAG in Western Blot, too. I used a high concentrated SDS-PAGE, but the signal was weak - but detectable...
I prefer to detect the FLAG expression using immunfluorescent staining. In your vector control you will get a signal in the cytosol while your untransfected cells should only show backgroundstaining.
Greetings,
Chakchel
I tried to detect 3xFLAG in Western Blot, too. I used a high concentrated SDS-PAGE, but the signal was weak - but detectable...
I prefer to detect the FLAG expression using immunfluorescent staining. In your vector control you will get a signal in the cytosol while your untransfected cells should only show backgroundstaining.
Greetings,
Chakchel
Hi Chakchel thanks for your reply and sorry for the late response. For my experiment, I can detect the Flag expression using Flag M2 Ab (sigma), whereas I can not detect the expression of my gene of interest using the antibody. which, in theory should come up at the same height of the Flag wstern blot. I have had no idea of this. There should be a Flag fusion protein which is expressed in my system, otherwise Flag should not appear on the gel at the exactly right height. So do you have some opinion on this.
Thanks a lot
Sorry I misunderstood the topic. Now disregard the flag control, I have got some Flag-tagged fusion protein screened for expression. As Flag antibody detected the stable expression of the fusion protein, the antibody specific for my gene of interst did not work (no expression appearing at the same height as that of the Flag), the antibody should be ok, as it can detect the endogenous expression of my gene of interest on the same gel. So now it seems difficult to explain that why I have the flag expression but not gene of interest expression. Has anyone come across the similar situation before? Could anyone give me some hint? Now I am trying to use another antibody for my gene of interest to see whether I can detect the protein. If it won't work, I may try to knock down my gene using siRNA and to see whether the Flag bands disappear (if do, it means my gene expressed finely)
I'm not sure if I fully understand the problem: on a western you can see a flag reactive band at the size you would anticipate your flag-tagged protein to run but you can not detect the flag-tagged protein using an antibody to the specific protein, even though you can detect the endogenous protein which runs right below the flag reactive band, yes? A simple explination (especially if your protein-specific antibody is a monoclonal) is that the flag tag has altered the availability of the epitope to the antibody. Sometimes a tag can really mess up a protein (functionally and structurally). Using another antibody is your best bet, however, it may be neccessary for you to move the tag or try a different one.
I agree with rk447...
Good luck!
Chakchel
Thanks for the answer, the antibody I used initially against my gene was polyclonal (I don't really know which epitopes it actually react with) and the new antibody I am trying now is the monoclonal one......so I don't know whether it's gonna work. I would belucky if so.
Actually, given some thought and the fact that your antibody is a polyclonal, I am most likely wrong. The gel you are running, I assume, is a SDS denaturing so epitopes should open up. Also, the polyclonal should have multiple epitopes it can identify so the chance of the flag tag messing with all of them is pretty much none. Sorry I can't be of more help. I hope your monoclonal works and this becomes one of the mysteries of science. Good Luck!
I am having the same problem. I overexpressed a flag/ha tagged protein and can detect very strong HA signal. When I used the protein specific polyclonal antibody, the signal is even weaker than untransfected or EGFP transfected cells. Has this anything to do with epitope available?