Two Dimensional Analysis of Human Sera by Chromatofocusing on Mono P™Followed by Fused Rocket Immunoelectrophoresis

Abstract

With the introduction of the Mono P™HR 5/20 prepacked column a new medium has become available for fast high resolution chromatofocusing of proteins in the pH range 4-9. The small bead size (10 μm) and monodispersity of the matrix (MonoBeads™) provide the basis for fast, high resolution separations without high operating pressures. The macroporous matrix allows separation of globular proteins with molecular weights up to 10 7 . The resolving power of chromatofocusing is comparable to that of isoelectric focusing. Since chromatofocusing on Mono P is technically simple and quick to perform the optimum pH interval for a given separation is easily obtained. Runs in broad pH intervals (2-3 pH units) at high flow rates can be used for fast screening and optimization. The pH interval of interest can then be expanded to obtain increased resolution. The preparative capacity of Mono P allows large sample loads to be run without loss of resolution. A single run thus provides enough material to proceed with a number of analytical and/or preparative methods. This combination of high resolving power and preparative capacity is illustrated here with the separation and subsequent analysis of some human blood plasma proteins.