Constitutive p42/44 MAPK phosphorylation - (Jul/04/2012 )
My lab frequently work with stimulation assays where we plate our cells, withdraw them from nutrient rich medium into charcoal stripped serum medium (lack of nutrients/growth factors etc) for two days. On the third day, we add a growth factor (such as EGF etc) to see if it induces phosphorylation of its receptor and subsequently its downstream proteins. Usually, in our control samples (to which we just add a vehicular control instead of the growth factor), we do not detect any phospho-akt or phospho mapk, however of late, we have been detecting very high levels of MAPK phosphorylation even in the control samples.
We believe that the causative factor could lie in our lysis protocol, which i've briefly outlined here:-
we wash our wells with cold PBS, before adding the RIPA lysis buffer (with added protease inhibitors) and then leaving the plate on ice for 30 minutes before scratching the plate with the tip of a pipette to wash off any remaining cells and centrifuging to separate the cell debris from the lysate.
It could be possible that when we wash the wells with cold PBS OR put the plate on ice before we add the RIPA, we induce cellular stress, which then triggers MAPK phosphorylation independent of growth factor receptor activation. We're yet to establish whether this is the reason for the excessive MAPK phosphorylation even in nutrient free media so I was wondering if anyone could possibly suggest any alternative explanation for this phenomenon?
I'm yet to analyze my samples in which i slightly tweaked the lysis protocol to utilize warm PBS instead of ice cold PBS.
Thank you for your help.
I dont think there is such huge changes when your cells are metabolically and physiologically inactive (on ice-cold PBS or ice bath).
Did you change the antibody you were using? Often polyclonal antibodies can have lot-to-lot variability.