Detecting phosphoproteins - different results when samples rerun (Jun/29/2011 )

Hi guys!

I´m trying to detect phosphoproteins, primarily ERK1/2, from rat brain tissue samples. I haven´t been able to induce an ERK phosphorylation with ligands that have previously been proven to do so in brain slice incubations. And believe me, I have tried

What I have noticed is that when I have rerun some samples I can get very different results... Previously I put pERK + tERK primaries to the same solution and chicken-anti-rabbit-ALEXA + rabbit-anti-mouse-HRP secondaries to the same solution to detect tERK with fluorescence and pERK with chemiluminescence at the same time from the same blot. Later I saw that it´s not a good idea; this compination can interact and give any kind of results.

Now I put first just pERK and later tERK. However, I still get different results of pERK after rerunning... also, some ligands that should INCREASE pERK seem to DECREASE it etc... argh!

Any suggestions?
I´m gratefull for all help!

induced phosphorylations normally exhibit typical kinetics which is not a linear increase but Koshland kinetics; so it is extremely important to have the kinetics of phosphorylation under control


don´t you think that parallel use of two antibodies may affect each other? the polyclonal may compete with the monoclonal for the same epitope?