I want to develop stable cell line for protein production, for that i linearized the plasmid containing Gene of Interest and Antibiotic Resistance gene for Geneticin and Transfected into CHO cells using Lipofectamine 2000 reagent as per the protocol. After 48 Hr's i subjected the cells to Geneticin Concentration and proceeded as per the protocol, Once Cells got adapted to 100 ug/ mL geneticin then i subjected to 200 ug/mL geneticin, likewise i subjected till 400 ug/mL Geneticin concentration, After that i subjected the cells for Protein production without adding Geneticin to the medium, After collecting the cell supernatant on day 5 and Subjecting to SDS-PAGE, I couldn't see any band which means my protein is not produced, I want to know How come the cells resistant to geneticin but not producing protein, What might be the possible reasons ?
This sentence lacks clarity "i subjected the cells for Protein production without adding Geneticin to the medium, After collecting the cell supernatant on day 5 and Subjecting to SDS-PAGE". What exactly did you do? Did you use lysate or conditioned medium?
My protein is extracellular, so i collected the supernatant and run the gel to look for Band.
I would start by doing Western blots of culture supernatants and cell extracts. SDS-PAGE alone may not be sensitive enough if there is low-level expression, and the protein may be getting stuck along the secretory pathway.
Hope it helps!
- Badges
- Science method