I am using cultured HEK293 cells and my readout for ERK1/2 activity is western blot using antibodies to pERK (pThr202/Try204) & pELK1 (Ser383).
Thanks.
PMA and EGF will preferentially go through ERK1/2. I use TNFa to activate all three ERK1/2,JNK and p38 and see a nice difference compared to EGF or PMA.
EGF or PDGF will activate ERk1/2 but not JNK. if you want to activate JNk UV( also will activate p38) or anysomicin
we also use PMA for ERK1/2 activation and gives pretty nice results as tested by inhibiting the pathway while stimulating.
50-100 nM PMA. You may want to perform a time course initially to determine the optimal time. Also do a basal point. Note that as you are using cultured cells, serum contains growth factors etc and so you should serum-starve the cells. Your lysis buffer should contain phosphatase inhibitors.
As suggested I've tried PMA, 25 nM for 5 mins (following 24 h serum starvation) in my HEK293 cells which gives me a robust & maximal phosphorylation of ERK1/2. However both Elk-1 antibodies are picking up multiple bands....I'll try a time-course of PMA next, upto 2 hours. Has anyone tried these Cell Signalling antibodies? Or had success IP-ing endogenous Elk-1? This is a control experiment for paper revisions so very little time for optimisation and any advice much appreciated.
Many thanks, Helen
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