Differential phosphoprotein labelling (DIPPL) using 32P and 33P.

Differential labelling techniques like differential in-gel electrophoresis (DIGE) enable mixing a control with an experimental sample prior to protein separation, thereby reducing complexity and greatly improving the resolution and analysis of changes in protein expression. Although the shift caused by phosphorylation to a more acidic pI can, in principle, reveal phosphorylation events using DIGE, analysis and verification of the phosphorylation are fraught with problems. Here we describe a differential phospho-labelling technique that obtains the same advantages as DIGE, which we named DIPPL, for differential phosphoprotein labelling. The technique involves labelling two samples, one with 32Pi (orthophosphate) and the other with 33Pi (orthophosphate). The two samples are mixed and proteins are separated on a single gel. Dried gels are exposed twice: once so that total radiation from 32P and 33P is collected on a film or screen; then acetate sheets are interposed between the gel and the screen such that 33P radiation is filtered out leaving 32P radiation to filter through. We demonstrate the utility of this approach by studying the MEK/ERK-dependent changes in stathmin phosphorylation induced by NGF in primary sympathetic neurons.