I am using a tetracycline-regulated Tet-off system (Doxycycline) to express my target protein in MKN28 and WT-A10 cells. I asking which is better to induce expression at culturing (0-time point) or wait until cells become 70% confluence and change medium into medium without doxycycline. Provided that cells start to detach when I follow the second way after 40 hrs even though induction should be continued for 96 hrs to obtain my protein.
Any suggestions/answer will be much appreciated.
Optimum time to see appreciable induction of a protein using Tet-on or Tet-Off system depends on the gene and cell-type. Therefore, the time needs to be optimized empirically. It may be time consuming, but needs to be done. See more details in our paper in the link below.
Link: http://cgd.aacrjournals.org/cgi/reprint/10/2/93
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