I am trying a transfection assay using HEK293 cells and wanna validate the transfection in flow cytometry. The plasmid contains the sequence of the protein and myc-DDK tag. Transfection is a transient introduction of the vector.
I can clearly see the successfully transfected cells as DDK+ cells; however, the antibodies against the supposedly induced protein does not detect it. Given that the following functional assay is consistent with the past study, I guess the protein is actually induced but somehow the antibodies fail to recognize it.
Is it possible that the antibodies recognize the antigen in primary cells (B cells, in my case) but not in some cell lines? Using polyclonal antibodies instead of monoclonal might help??
Your antibody may fail to detect the transfected protein in HEK293 cells due to epitope masking by the DDK tag, altered protein folding or post-translational modifications, or monoclonal antibody specificity. HEK293 cells may lack modifications present in primary B cells, affecting epitope recognition. Using polyclonal antibodies, testing different fixation methods, or confirming expression via Western blot can help troubleshoot the issue. Ken Yasaka
Dear Ken Yasaka
is the monoclonal that you are using predicted to bind an extracellular region of the protein?
best
Manuele
Muhammad Mubashir Thank you! That makes sense.
Manuele Martinelli Yes, the antibodies that I currently use are the monoclonal ones reactive to the extracellular domain.
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