An immunoblotting method for high-resolution isoelectric focusing of protein isoforms on immobilized pH gradients

Post-translational modifications such as phosphorylation and acetylation ar e important elements for regulating the activity of enzymes or structural p roteins. These modifications give rise to isoforms that are often not resol ved by separation methods relying on the size of proteins. Here, we optimiz ed an isoelectric focusing (IEF)-immunoblotting method suitable for analyzi ng protein isoforms in total cell extracts. The separations were carried ou t in parallel on commercially available immobilized pH gradient slab gels ( IPG). The buffer used for separation contained urea, thiourea, dithiothreit ol, as well as the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propa nesulfonate (CHAPS), and was designed to match those used in two-dimensiona l poly acrylamide gel electrophoresis (PAGE) separations where efficient so lubilization is required. Proteins were transferred to membranes by passive diffusion in the presence of 4 M guanidinium chloride using protocols opti mized for several protein classes (tubulin, stathmin, 14-3-3 proteins) some of which required removal of CHAPS prior to transfer. In conjunction with narrow-range pH gradient gels, excellent resolution of isoforms differing b y phosphorylation or acetylation was achieved. The usefulness of pi and tit ration curve calculations for predicting the pi shifts expected for post-tr anslational modifications of proteins with known amino acid composition was demonstrated. Using stathmin - which contains four phosphorylation sites - as an example, the effects on the pi-shifts were well predicted. This sens itive and widely applicable IEF-blotting technology is expected to be espec ially suited for analyzing protein isoforms first detected by two-dimension al electrophoresis.