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.
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