In article <2vitui$q7q at dingo.cc.uq.oz.au>, forrest at biosci.uq.oz.au (acer) writes:
> G'day all,
> Does anyone know of a NON-radioactive procedure for testing whether a
> protein is phosphorylated or not? We are interested in whether our recombinant
> is being phosphorylated when expressed in baculovirus. We've thought about using
> P32 in the culture medium but this is a messy and dangerous technique, does
> anyone know of a non-radioactive technique for determining whether our protein
> is phosphorylated?
Yes there is. By means of mass spectrometric analysis of your expressed
protein, we can see how many phosphorylations are on your protein. The phosphate
group gives an extra mass shift of 80 Da. This to start with.
Additionally with non-labelled P32. We can also determine where the phosphates
and other modifications (?) are sitting in your protein. This takes more time
but can be done also on the mass spectrometers.
[This procedure was already performed 3 times in the last months here at the
EMBL.]
Good luck for furter thinking.
Tony Houthaeve
