Overexpression plasmid is not expressing protein - (Apr/01/2011 )

OK, I did that, got an additional band in the sample that was transfected only with my plasmid. Again my GFP sample and GFP,plasmid sample had no difference. Can anybody explain this to me?

I am probing against actin by now to confirm there is a difference in band intensity.

Hi,

I am trying to over express a demethylase protein in a lung cancer cell line but my cells are refusing to express the protein.

I know the transfection is good because I have co-transfected my CMV-KDM (lysine demethylase) plasmid and CMV-GFP into the lung cancer cell line. The GFP was detectable by flow cytometry and western blot, therefore the cells were transfected.
I know the plasmid is good because I have had it sequenced and it also over expresses at an mRNA level, validated by qPCR, in my lung cancer cell line.
I have also overexpressed this plasmid in HeLa and probed for anti-flag and detected the protein in HeLa.

So the lack of protein expression is cell specific. So far I have transfected HeLa which do express the protein, and 2 lung cell lines which do not express the protein.

I am super excited that it might be a lung cancer phenomenon. I am looking at other cell types to make sure first.
But I have no idea where to go from here. There are many possibilities of mechanisms that can be inhibiting translation (the mRNA is expressed but the protein is not).
Can you please make suggestions of what and how I can look at possible translational inhibitors.

Much appreciated.