What kind of protein is it?  How did you check the protein expression? Some proteins could require a signal peptide to be expressed for instance on the plasma membrane or in some membrane intracellular compartments...

thanks Ilaria for your comment. it is viral envelope protein and I checked the expression with ELISA using different sets of antibodies that normally recognize this protein. Actually, I followed the protocol established in the lab for such purpose and the positive control construct was developed in the same way. 

Hey Ahmed, I think factor like temperature and incubation period after induction can be quite critical for protein expression. Some protein are quite sensitive to temperature changes, so if your temperatures is slightly altered, the protein might not be able to expressed. Also, you can try to incubate your protein needs overnight to get a better yield. 

Hope it helps.

Maybe you can check the presence of DNA and mRNA expressed in the cells by PCR first.

How do you ensure DNA precipitates are forming??? Did you do a mock to see precipitates form with Clacium pHosphate without DNA ?? do they form too quickly and big????

Do you see the plate too often??? after transfection??

Also, are you using a fresh lot of 293T or just a longly subcultured/passaged line.... BTW whats the positive control/.. you mentioned???

CELL LYSATE??? WHAT DO YOU MEAN..... are you discarding supernatants???  

Consider all above...

The cell line you are using is temperature sensitive and you can also try 293EBNA cell line

Dear Kumar, 

Can you please elaborate on the temperature sensitive part of 293T cells? 

thank you all for your valuable comments. in regards to the positive control, I used the same vector but encoding a certain protein and I used it in parallel for transfection and detection of antigen by ELISA. the ELISA gave positive results with the cell lysate of the positive control while the lysate of cells transfected with my gene gave negative results. I think this means the gene was not expressed although the sequencing results says it is in frame with the promotor.

is there any possibility for a mutation within the plasmid that affect the expression?

Hi Ahmed,

Why don't you check other methods for checking the expression. If you still have the cell lysate why don't you try WB? In addition, did you sequence the plasmid in hand yourself? or the plasmid and the sequencing results were passed to you from someone else? if this is the case, please sequence the plasmid in hand yourself. 

Ahmed, interested to learning if you have solved this issue?  Any progress to share with us?

We are trying to express a viral protein with pcDNA in 293A and 293T, with no protein expression at al.  

Thanks, Andrew 

Now, I found the same problem. How do you fixed the problem?

I hope your experience can help me

Thank you so much

Dear All,

Thanks All and sorry for late response. The problem was related to the plasmid itself. A new preparation of the plasmid did the job. The old one I propagated in bacteria to make big batch, some mutations might be accidentally introduced which later negatively affected the expression.

good luck all