Natalia, I am a bit confused. Tet-On will activate expression in presence of Doxycyclin only, Tet-Off will activate expression in absence of Doxycyclin only.

I.e. if you are using a Tet-Off, adding Doxycyclin will actually shut off transcription. In that case, our targets usually lose the bulk of mRNA after 5 hours, and protein is undetectable after 6 hours by Western.

I don't know however if the inverse is true for induction using Tet-On, as you seem to be doing.

Hello Wolf

Thank you for your message. I am using Tet Off, so I induce expression by removal of Dox from the medium. Induction itself is visible on RNA level by Northern blot, but only a portion of cells is induced. Is it a way to increase the number of cells induced by removal of Dox?

Thank you very much.

Actually I have zero experience doing it this way around, but my best shot would be to administer several rounds of washing cells in fresh YPD, interrupted by stirring and mild centrifugation (

Hi Natalia,

I have some experience with integrated tet-Off promoters in yeast. I do understand that you see induction on a northern blot, so I wonder how do you know that some cells are activated while some others aren't? Do you have a Fluorescent protein regulated by such promoter?

If so, it happens very frequently that Gfp is visible only in some of the cells in a population, the reason for this not being entirely clear. Among other things, the folding rate and the half-life of your protein may also play a role here. In my hands, 6 hours was enough for induction, but I never checked at subpopulation levels

Hi Alessandro and thank you for your message. I use fluorescent in situ hybridization to look at the individual cells.

thus it's really at the mRNA level. Amazing.

Increasing the quantity and quality of washes to remove Dox fully, as suggested by Wolf, is supposed to help. If it doesn't, I wouldn't know what else to suggest. Good luck

Hello Natalia,

I worked with Tet-on or degron system but never with tet-off. If your population is not synchronized and the protein you tried to induce cell cycle dependent maybe it explain that you see induction only in some cells, the one at the rigth cell cycle phase during removal of doxycycline. How long to you leave cells in doxycycline before removal? I will suggest to do a shorter time of culture before and washes after. You can as well synchronize the cells to avoid cell cycle effect if you want to. Good luck