I have found Sigma's anti-FLAG M2 antibody to be very good for immunofluorescence, and you say that there is signal by Western, suggesting that protein is being made. To troubleshoot this, I wonder what the signal looks like in the ~5% of cells where you see it? If it is very dim, then you may be near the threshold of detection, and more cells are transfected than you're able to see. If this is the case, you may need to increase your concentration of primary or secondary antibody, or increase your exposure time. Or, if your cells don't have much more signal than the background, you may need to wash more extensively. Also, what do you know about the protein? Does its localization change? If a protein is clustered (in protein-complexes, or localized to a specific organelle) it will appear much brighter than if it is diffuse across the cytoplasm or plasma membrane. Is it possible that you're not seeing signal because generally the protein is dispersed, and only rarely clustered? In this case, you will have to make sure to keep all settings (exposure time, etc) the same for all images, and do contrast manipulations exactly the same for all images, and compare transfected with untransfected cells (you should be doing this anyway). You may only be able to see a subtle difference this way. One final thought would be about cell permeabilization - since GFP is epifluorescent, you would be able to see it in cells with intact membranes. But if your membranes are not permeabilized, antibody will not get inside the cell, except cells that have had some damage from the fixation, which could easily be ~5%. It's hard to know for sure what's going on - but these are some issues I've run across before, hope it helps!

Thanks a lot David for the answer.

I have tried to increase antibody concentration (both primary and secondary) but still didn't see much of my flag tag protein. It is located in the nucleus and I see another protein in the nucleus (endogenous) very well so i assume the permeabilization worked. The GFP was served only for transfection control i didn't use those cells in the immunofluorescence assay. Playing with the exposure time didn't help :( .

did u use 3x flags or one? I use one

It looks like you're using good controls, how frustrating.

I used one FLAG tag. There's also a difference between the M1 and M2 antibodies, where M1 can only recognize FLAG on the N-terminus, and M2 can recognize it in other contexts. I'm guessing this isn't the problem, but it's all I can think of.

Is there a difference between the conditions you use for the GFP transfection and your FLAG-construct transfection? If you try co-transfection, the GFP+ cells should also have taken up your FLAG-protein construct, so that at least you're internally controlled for the same experiment, otherwise, I'm sorry, I can't think of what to try.

I'll try to do co-transfection its a good idea.. Thanks for the brainstorming. if you have more ideas  I'll be grateful

You mention your tagged protein localizes in the nucleus normally. Did you see there? Probably the transport does not work normally or weak because of tagging... 

Hi I agree with Attila. How about trying an inducible expression construct? Since it is a nuclear protein, the tag might be interfering with its function. Also, transient transfections can have a huge variation on their efficiencies. Perhaps an inducible stable might overcome the problem? One more suggestion will be to tag it with a yfp if you are just doing a preliminary localisation study. Good luck!

Attila and Adam, thanks for the bright comments. I have decided to produce stable cell line which will make things easier in futuer experiments. hope it will also improve my immunofluorescence assay.