Hi all,
I am going to perform immunofluorescence analysis of transfected HeLa cells to determine the localization of the protein of interest.The protein is tagged with GFP and I am using Doxycycline (1ug/ml) to induce the expression of GFP tagged protein in the cells.I would like to know whether 24 hours would be enough to induce the expression of my protein or should I incubate longer (48 hours)?.I am also adding Nocodazole to the cells to induce mitotic cell cycle arrest after Dox induction.Kindly confirm whether I have to aspirate the media containing Dox, wash with PBS and add fresh media before the addition of Nocodazole?
Thank you .
24 hours of Dox is enough to induce expression (just make sure cells are not too confluent and everything is added properly, e.g. when you add Dox to the medium, make sure to shake the plate in your hands a little to distribute it evenly).
The only thing I need to warn you about that GFP protein takes time to mature after expression start, and the time of maturation is about 24 hours - so, if you try to observe the cells exactly 24 hours after Dox, it may not be enough for GFP signal to appear (no green cells). Wait for 30-36 hours for the signal, 48 hours would do, too.
About Nocodazole, IMHO either way is ok. I wouldn't aspirate it, Dox and Nocodazole don't interact.
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