I'm investigating the phosphorylation of a ~130 kDa protein that is difficult to express (due to GC rich nature of the gene), and difficult to immuno-precipitate large amounts of.
I have tried radiolabelling with hot orthophosphate, however, although IP works, I have had no success due to the high background (if anyone has suggestions to improve this approach please feel free to suggest).
I'm also contemplating in vitro labelling, however, in my context in vivo phosphorylation would be the best.
I'm contemplating the use of ProQ diamond because if it works according to the manufacturers suggestions, it would eliminate the need for whole cell labelling with 32P. Currently, in my endeavours to identify the phosphorylation site of this protein, I'm employing a mutagenesis and labelling approach.
Any feedback/discussion in regards to ProQ diamond, or any of the approaches discussed in this message would be appreciated.
Edited by ProtoXpert, 13 January 2005 - 12:05 AM.