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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

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

-Inmost sun-