Unbreakable Complexes in SDS-PAGE/Western Blots?

Wolfgang Schechinger novalidaddress at nurfuerspam.de
Tue Jun 20 08:24:13 EST 2006


Dear Experts,

I am detecting the phosporylated form of a protein by western blotting.

Besides the band at the desired/expected MW (170kD), I also get a clear
signal (which under some conditions is even stronger than the actual
band of interest) at an apparent size of approx 250kD. And I see a
clear time course of this band in a stimulation experiment. It is
unlikely that this band is something like an unprocessed or partially
processed pre/pro form. I rather suspect that there is a complex with
some other (yet unknown) protein. I see this band with several
antibodies against different serine phosphorylations of my protein of
interest as well as with pY20 and total protein. I am not satisfied
with the usual parroted explanations about precipitates, artifacts
etc., but actually I expected that after boiling (7.5 min / 99degC)
with Laemmli (BME) all non covalent interactions should be broken.

I am looking out now for suggestions on how to prove my hypothesis with
a simple experiment (no mass spectrometry etc at this stage please) ,
i.e. how to demonstrate that there is a complex. So, how may I disrupt
it?

All input is welcome!

Wolfgang



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