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!
I´m gratefull for all help!
Edited by rajah, 29 June 2011 - 06:40 AM.