Transfection and Expression: getting transcript but no protein expression - (Nov/16/2006 )

Hi all

I am having trouble with detecting a protein that should be expressed by transfected HEK cells.

I did RT-PCR with specific primers for my gene of interest and I have clear bands (no bands in untransfected HEK 293 cells). So my gene is in the cell and even gets transcribed, but i cannot see any protein in western blot!

I cloned a CD33 secretion signal peptide in front of the protein sequence, so secretion should be initiated.

For SDS-Page and subsequent blotting, I collect supernatant and also prepare cell lysate and concentrate about 20fold using centricon concentrators. I then use anti-his antibody (sequence of the plasmid was checked and presence of his-tag verified) and protein-specific antibody to detect my protein, but i cannot see any specific bands!

Can someone give me a tip?

thanks al lot, matt

There are many things that could be an issue here. Just a start: Check open reading frame sequence. Is the protein stable (may want to use protease inhibitors). Could be an issue with splicing if there are splice donor/acceptor sites in the transcript.

Hi tap

thanks for your reply.

The protein should be stable. Nevertheless, I added protease inhibitor while harvesting the supernatant and preparing cell lysate.

Open reading frame is correct. splicing sites should not be present.

My boss said it is normal that you dont see protein expressed from a 10cm plate if the culture is still mixed clones and not monoclonal. I should first prepare about 20 clones, than check them for mRNA with RT-PCR and only then harvest supernatant from a 10cm plate of my clonal cells. Seems counterintuitive to first prepare clones and only then check protein expression, isn't it?

It depends on the promoter you are using as well as how long after transfection you are assaying. A strong promoter should give easily detectible expression up to 72 h post-transfection.