Phospho Protein detection method - Phosphoprotein detection with phospho-specific dye (Jan/12/2005 )
Does anyone have any experience using phospho-protein specific stains such as ProQ diamond (from molecular probes (invitrogen)).
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.
Colorimetric stains for inorganic P have not gotten any better from the 1929 method of Fiske/Subbarow. I suggest in-vitro 32P labeling. It is the only method that will be ultimately satisfactory