Phosphoprotein detection on protein electroblot using a phosphate-specific fluorophore.

Abstract

The reversible phosphorylation of phosphoproteins is a vital regulatory process for many cellular pathways. A reliable and simple fluorescent detection technique for phosphoproteins has been developed using a small-molecule organic fluorophore, Pro-Q Diamond dye. This was originally developed for use in gel staining, but a new formulation has allowed for its use in protein blotting. The dye binds noncovalently and selectively to the phosphate moiety, so proteins lacking phosphate groups and other macromolecules such as DNA or RNA are not detected. It uses a standard electrophoresis and electroblotting technique, which can blot the sample onto nitrocellulose membranes or polyvinylidene fluoride (PVDF). The electroblotting is followed by staining with the dye and destaining. The blot can then be read by multiple types of imaging devices such as a laser-based gel scanner. This process is compatible with matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) and Edman sequencing. It can also be followed by standard chemiluminescent, colorimetric, and fluorogenic detection techniques used in immunoblotting.