I recently noticed a problem in my transient transfection. I co-transfected monoclonal antibody heavy and light chain expression vectors into 293T cells and harvested the supernatant to assess them in ELISA. My transfection used to be so efficient with a high level of antibody production but recently I noticed that the antibodies can not be detected by ELISA and Western blot. Since the routine secondary antibody was discontinued to be produced, we had to use another secondary antibody in ELISA and Western blot. So, I assumed that my problem is due to the change of secondary antibody. However, trying other secondary antibodies I finally found that the transient transfection did not result in any monoclonal antibodies expression. So, I guess something is off in the expression system but am not sure what that could be. Worth to mention that the quality and sequence of the plasmids ae great.
In this regard, I was wondering what could be the transient transfection efficiency difference between HEK293 versus HEK293T cells if my antibody expression vector has SV40 late polyadenylation signal? I guess maybe my cells are not really 293T cells!! I got them from a colleague and guess she gave me HEK 293 instead of HEK 293T cells by mistake!!
Your help is appreciated in advance.
There were no major differences in transfection efficiency between lines 293 and 293T. Most expression plasmids give an equivalent result.
However, integration of the T antigen into the 293T line significantly improved the expression of the plasmids having the SV40 promoter.
Nevertheless, since the 293T line has also incorporated the neomycin resistance gene, this cell can not be used with G418 to increase the expression of the protein of interest.
Thanks Jean and Aim. It was so helpful. I will try your suggestions and hopefully this time it works.
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA