Hello, I am puzzled with my protein immunoblotting. I have constructed a plasmid pcDNA3.1(+) concluding my target gene fusion with FLAG tag on its N-terminal sequence. We got the plasmid sequence and detected the correct expression for both FLAG tag and GENE protein expression for twice.

However, after I repeated twice experiment, I transfected the plasmids for the third time, I could not detected FLAG expression anymore. I have checked the sequence because there was always the same tube plasmid I used. And I can alwasys detect the GENE expression by specific antibody, but the FLAG tag can not be seen anymore.

I am very puzzled with this problem, for there is nothing changed all the time. What's more, we can not detected the protein FLAG tag expression, interestingly, we found the specific lane using FLAG antibody in our IP samples(with FLAG beads). Honestly, my target protein is big (about 250kD), and the only reason I can suspect now is the huge protein shelter my FLAG tag.

Is there anyone can help me or give any advise?

Thanks much.