I have cloned five bacterial genes into pFLAG-CMV-6C (Sigma) vector along with two human genes in the same vector. Then I over-expressed these clones into HEK293T cell and lysed the cells after 30 hrs. of transfection to make a whole cell lysate. In western blot with FLAG-HRP antibody I am getting only one band. The band I am getting is the human gene expressing, with the rest of all the clones not expressing at all.

I have also checked these parameters:

1. Cloning- All genes are in frame into vector and 100% identical sequence.

2. plasmid DNA- 260/280 ratio is 1.9 so it seems like good quality DNA. I also ran the DNA into agarose gel.

After all I have repeated this experiment 2 times and I am stuck for all my future plans. Please give your kind suggestions to resolve this issue?

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