Two-dimensional micro-separation technique for proteins and peptides, combining isoelectric focusing and gel gradient electrophoresis.

The two-dimensional combination of gel electrofocusing and gel gradient electrophoresis yields separate information about differences in net charge and molecular weight of macro-ions. A micro-version of this technique is described. The first dimension electrofocusing run is performed in cylindrical polyacrylamide gel of capillary size, using xylene cyanole FF as a reliable marker dye to indicate the final separation stage when proteins reach their isoelectric points. In the second-dimension run the focused proteins are separated in a continuous slab gel gradient of less than stamp size with total acrylamide concentration ranging from ca. 1% to more than 40%. Spreading of peaks in the gradient slab gel is shown to be related to the approach of protein to its pore limit in the gel. This spreading is a useful indicator of the final position of a protein in the gel if molecular weights are estimated according to the separation pattern in the gradient gel. The method described has been developed for the separation of proteins extracted from large single cells (i.e., neurons of the mollusc Aplysia californica.