I have an in trans assay where I take conditioned media from transfected 293T cells and treat MG-63 cells with the conditioned media.
The problem I'm having is three of my controls (untransfected conditioned media, serum-free media (DMEM), and vector control have increased levels of phosphorylated activity in some experiments.
I serum starved my MG-63 cells for 24hrs prior to treatment. I also treat my cells with the conditioned at room temperature instead of 37C for 2hrs.
I'd like to know if serum starvation (0% serum) for 24hrs is too long and also if room temperature treatment with soluble protein is too harsh for the cell.
We ususally run our phosphorylation assays for 3h at 37° (plus over night sarvation before treatment) but add 0,1% FBS.
The serum starvation itself will induce phosphorylation so the appropriate controls are extra important.
- Badges
- Science method