Dear all,
I have tried to use a mammalian Expression Vector System to overexpress a protein of interest in AGS cell line. Cells were selected based on puromycin resistance conferred by successful vector integration. However, despite successful antibiotic selection, I haven’t detected overexpression of the desired protein in this cell line. This has now happened for two different vectors encoding different target proteins.
Does anyone have any tips to reinforce the expression of the desired proteins or a possible explanation for this?
Thank you in advance,
Andreia Peixoto
Could be many reasons. Are you sure the coding region is intact, that the promoter is active, or that your antibody is good enough and specific enough? What kind of vector is this, plasmid or lentivirus? Have you tried transient expression just to see if you get anything at all? How do you prepare protein lysates? Is your protein insoluble or chromatin associated? So many reasons....
Hi Andreia Peixoto, I totally agree with Gregory Dressler. Could be useful to test in a transient transfection the overexpression of your POI just to check the functionality of your vector and promoter of choice.
Then what came to my mind since you said it happened twice with 2 POI in the same vector could be a vector-related issues.... Could it be that only the Puromycin expression cassette got integrated? Are there some highly homologous sequence surrounding it? It would be useful to have more info about the vector you are using...
Let us know, I'm getting curious! Good luck!
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