I made a stable transfected HEK 293 cell line to expressing my protein, and now I want to use the protein containing supernatant(FCS free) to stimulate the normal HEK 293 cell again, to see whether my protein has the phosphorylation effect and then check it with Western.
I starved the normal HEK cells for 24hours and the add the supernatants, but I also got some bands in the negative group(which one add FSC free medium) and also some bands in the Vector group( which contain the Vector cell supernatants)
So is something wrong here? the time for the starvation time is not long enough or i need to use some other medium?
All medium I used is DMEM from gibco.
Thanks.
Perhaps the starvation is triggering something? Do you think it is worthwhile using something like optimem instead of FCS free media?
24h starvation is sufficient to almost abolish any ERK-p.
Did you wash your cells with PBS or DMEM (no FCS) after removing media with serum?
Some cells get stressed when one removes media and replaces it with new media which can lead to increased phosphorylation; but I never seen that with HEK cells.
Hi Tengfei,
24h starvation is enough to strongly decrease ERK phosphorylation. Since I think a bit more (up to 48h) may be better if your cells will not be defficient for anything in these conditions.
You may obviously wash your cells with FCS-free medium before treatment since FCS may have delayed effects. Also the medium could have... (phenol red by example).
Another thing to take into account is that the composition of your "vector cell medium" is different from the control medium, as cells used part of the nutriments in the first one. This may be of less importance if you test ERK phosphorylation at very short times of exposure (1-30 or 60 min) which is classical.
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